Portrait of Dr Richard Williamson

Dr Richard Williamson

Senior Lecturer in Protein Biochemistry
Undergraduate Admissions Officer
Faculty Director of Recruitment and Outreach

About

Richard Williamson studied Biochemistry as an undergraduate at the University of Bath and then moved to the University of Kent in 1986 to study for a PhD with Professor Robert Freedman. The work investigated the structure and function of tissue inhibitor of metalloproteinases (TIMP), the natural protein inhibitor of a family of proteinases responsible to tumour metastasis and connective tissue damage in arthritis.
After 6 years of post-doctoral work at Kent and Celltech Ltd. (Slough), Richard was awarded a 5-year Research Fellowship in 1996 funded by the Arthritis Research Campaign. Work during this time lead to the determination of a 3-dimensional structure for TIMP-2 by NMR and mapping of the proteinase binding site on the TIMP-2 structure.
Richard became a Lecturer at the University of Kent in 2001 and a Senior Lecturer in 2008. Research interests broadened during this time to include protein disulphide isomerase (important for the correct folding of proteins in the endoplasmic reticulum) and structural analysis of mycobacterial proteins known to be important for M. tuberculosis infection.
Richard has served as Undergraduate Admissions Officer for the School of Biosciences since 2011 and as Faculty Director of Recruitment and Outreach since 2017. Richard is the current chair of the External Relations Committee.

Research interests

Protein structure and function. Specifically protein folding both inside and outside of the cell, and renaturation of proteins from bacterial inclusion bodies. The use of NMR to study protein structure and dynamics.

Teaching

Undergraduate 

  • Biological Chemistry – BI321/BI3210/BI322/BI3220 
  • Skills of Bioscientists 2 – BI532
  • Proteins: Structure and Function – BI629 (module convenor) 

Postgraduate 

  • Advanced Analytical and Emerging Technologies for Biotechnology and Bioengineering – BI852

Publications

Showing 50 of 59 total publications in the Kent Academic Repository. View all publications.

Article

  • Freedman, R. et al. (2017). ‘Something in the way she moves’: The functional significance of flexibility in the multiple roles of protein disulfide isomerase (PDI). Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics [Online] 1865:1383-1394. Available at: https://doi.org/10.1016/j.bbapap.2017.08.014.
    Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI.
  • Richards, K. et al. (2016). Combined ligand-observe 19F and protein-observe 15N,1H-HSQC NMR suggests phenylalanine as the key ?-somatostatin residue recognized by human protein disulfide isomerase. Scientific Reports [Online] 6:19518-19526. Available at: http://doi.org/10.1038/srep19518.
    Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (Kd) of the peptide-ligand ?-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using 19F ligand-observe and 15N,1H-HSQC protein-observe NMR methods. Phe residues in ?-somatostatin are hypothesised as important for recognition by hPDI therefore, step-wise peptide Phe-to-Ala changes were progressively introduced and shown to raise the Kd from 103?+?47??M until the point where binding was abolished when all Phe residues were modified to Ala. The largest step-changes in Kd involved the F11A peptide modification which implies the C-terminus of ?-somatostatin is a prime recognition region. Furthermore, this study also validated the combined use of 19F ligand-observe and complimentary 15N,1H-HSQC titrations to monitor interactions from the protein’s perspective. 19F ligand-observe NMR was ratified as mirroring 15N protein-observe but highlighted the advantage that 19F offers improved Kd precision due to higher spectrum resolution and greater chemical environment sensitivity.
  • Sorge, J. et al. (2015). Q2DSTD NMR deciphers epitope-mapping variability for peptide recognition of integrin ?v?6. Organic & Biomolecular Chemistry [Online] 13:8001-8007. Available at: http://doi.org/10.1039/c5ob01237f.
    Integrin ?v?6 is a cell surface arginine-glycine-aspartic acid (RGD)-specific heterodimeric glycoprotein that is only expressed on epithelia during processes of tissue remodelling, including cancer. The specificity and molecular nature of interactions toward this integrin are poorly understood and new insights into such processes are important to cell biologists and pharmaceutical drug discovery. This study demonstrates the application of quantitative two-dimensional saturation transfer (Q2DSTD) NMR to obtain precise details of peptide interactions with integrin ?v?6 and their correlation to specificity for the integrin. This approach highlights subtle but significant differences in ligand contact by three related 21-mer peptides: FMDV2, an ?v?6 specific peptide and DBD1 and LAP2T1 peptides that bind many ?v integrins in addition to ?v?6. FMDV2 and DBD1 differ only by the cyclisation of DBD1; a process that removes ?v?6 specificity. Q2DSTD NMR demonstrates these peptides experience significantly different interactions with the integrin; FMDV contacts primarily through four residues: 6Leu, 10Leu, 12Val and 13Leu, whereas DBD1 and LAP2T1 have more widespread contacts across their sequences. Q2DSTD NMR combined two-dimensional STD with quantitation by considering the relaxation of the ligand (CRL) to provide precise ligand contact information. This study also examines the role of CRL in the Q2DSTD process and how quantitation modifies STD data and unravels epitope-mapping variability to provide precise results that differentiate interactions at the atomic level for each peptide.
  • Curtis-Marof, R. et al. (2014). 19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b?x from human protein disulphide isomerase (hPDI). Organic & Biomolecular Chemistry [Online] 12:3808-3812. Available at: http://dx.doi.org/10.1039/c4ob00699b.
    We report a protein-observe 19F NMR-based ligand titration binding study of human PDI b?x with ?-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single 19F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 ?M.
  • Curtis-Marof, R. et al. (2014). 19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b'x from human protein disulphide isomerase (hPDI). Organic & Biomolecular Chemistry [Online] 12:3808-3812. Available at: http://dx.doi.org/10.1039/C4OB00699B.
    We report a protein-observe (19)F NMR-based ligand titration binding study of human PDI b'x with ?-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single (19)F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 ?M.
  • Ley, N. et al. (2014). Optimising selective excitation pulses to maximise saturation transfer difference NMR spectroscopy. RSC Advances [Online] 4:7347-7351. Available at: http://dx.doi.org/10.1039/C3RA46246C.
    A simple method is presented that optimizes the STD NMR Gaussian pulse to deliver significant increases in STD amplification factors with minimal perturbation of the ligand. This approach is practically demonstrated using the wheat-germ agglutinin/N-acetyl-D-glucosamine protein–ligand system.
  • Irvine, A. et al. (2014). Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway. PloS one [Online] 9:e82511. Available at: http://dx.doi.org/10.1371/journal.pone.0082511.
    In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10(-5) M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding - differential affinity, rapid ligand exchange and conformational flexibility.
  • Amin, N. et al. (2013). High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility. Biochemical Journal [Online] 450:321-332. Available at: http://dx.doi.org/10.1042/BJ20121635.
    ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxin-fold domains homologous with the non-catalytic b and b' domains of PDI. The structure in solution of the N-terminal b-like domain of ERp27 was solved using high-resolution NMR data. The structure confirms that it has the thioredoxin fold and that ERp27 is a member of the PDI family. N-15-NMR relaxation data were obtained and ModelFree analysis highlighted limited exchange contributions and slow internal motions, and indicated that the domain has an average order parameter S' of 0.79. Comparison of the single-domain structure determined in the present study with the equivalent domain within full-length ERp27, determined independently by X-ray diffraction, indicated very close agreement. The domain interface inferred from NMR data in solution was much more extensive than that observed in the X-ray structure, suggesting that the domains flex independently and that crystallization selects one specific interdomain orientation. This led us to apply a new rapid method to simulate the flexibility of the full-length protein, establishing that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker, consistent with the NMR data.
  • Taylor, S. et al. (2013). Measuring protein reduction potentials using 15N HSQC NMR spectroscopy. Chemical Communications [Online] 49:1847-1849. Available at: http://dx.doi.org/10.1039/c3cc38952a.
    NMR spectroscopy was used to measure reduction potentials of four redox proteins by following multiple 15N HSQC protein resonances across a titration series using mixtures of oxidised and reduced glutathione. Results for PDI a, PDI ab and DsbA agree with the literature and our result for ERp18 confirms this protein as an oxidoreductase of comparable or greater reducing strength than PDI a.
  • Wagstaff, J. et al. (2012). NMR relaxation and structural elucidation of peptides in the presence and absence of trifluoroethanol illuminates the critical molecular nature of integrin avb6 ligand specificity. RSC Advances [Online] 2:11019-11028. Available at: http://dx.doi.org/10.1039/C2RA21655H.
    Integrin avb6 is an important emerging target for both imaging and therapy of cancer that requires specific ligands based on Arg-Gly-Asp (RGD) peptides. There remains little correlation between integrin-RGD ligand specificity despite studies suggesting an RGD-turn-helix ligand motif is
    required. Here, we describe the application of 15N NMR relaxation analyses and structure determination of avb6 peptide ligands in the presence and absence of trifluoroethanol (TFE) to identify their critical molecular nature that influences specificity, interaction and function. Two linear
    peptides; one known to demonstrate avb6 specificity (FMDV2) and the other based on a natural RGD ligand (LAP2), were compared to two additional peptides based on FMDV2 but cyclised in different positions using a disulphide bond (DBD1 and DBD2). The cyclic adaptation in DBD1
    produces a significant alteration in backbone dynamic properties when compared to FMDV2; a potential driver for the loss in avb6 specificity by DBD1. The importance of ligand dynamics are highlighted through a comprehensive reduced spectral density and ModelFree analysis of peptide 15N
    NMR relaxation data and suggest avb6 specificity requires the formation of a structurally rigid helix preceded by a RGD motif exhibiting slow internal motion. Additional observations include the effect of TFE/water viscosity on global NMR dynamics and the advantages of using spectral density NMR relaxation data to estimate correlation times and motional time regimes for peptides in solution.
  • Schmidt, J. et al. (2011). One-bond and two-bond J couplings help annotate protein secondary-structure motifs: J-coupling indexing applied to human endoplasmic reticulum protein ERp18. Proteins: Structure, Function, and Bioinformatics [Online] 79:428-443. Available at: http://dx.doi.org/10.1002/prot.22893.
    NMR coupling constants, both direct one-bond (1J) and geminal two-bond (2J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18-kDa protein comprise 1268 values of 1JCaHa, 1JCaCb, 1JCaC', 1JC'N', 1JN'Ca, 1JN'HN, 2JCaN', 2JHNCa, 2JC'HN, and 2JHaC'. Comparison with 1J and 2J data from reference proteins and pattern analysis on a per-residue basis permitted main-chain f,y torsion-angle combinations of many of the 149 amino-acid residues in ERp18 to be narrowed to particular secondary-structure motifs. J-coupling indexing is here being developed on statistical criteria and used to devise a ternary grid for interpreting patterns of relative values of J. To account for the influence of the varying substituent pattern in different amino-acid sidechains, a table of residue-type specific threshold values was compiled for discriminating small, medium and large categories of J. For the 15-residue insertion that distinguishes the ERp18 fold from that of thioredoxin, the J-coupling data hint at a succession of five isolated type-I b turns at progressively shorter sequence intervals, in agreement with the crystal structure.
  • McVicker, G. et al. (2011). SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12. Journal of Biological Chemistry [Online] 286:32026-32035. Available at: http://dx.doi.org/10.1074/jbc.M111.266619.
    We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (OSA1 and OSA2), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap OSA1 and OSA2, respectively. Mutations that disrupt either OSA1 or OSA2 eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ?1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.
  • Wagstaff, J., Howard, M. and Williamson, R. (2010). Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin avß6 binding peptides for NMR. Molecular BioSystems [Online] 6:2380-2385. Available at: http://dx.doi.org/10.1039/c0mb00105h.
    The integrin ?v?6 is up-regulated in several cancers and has clinical potential for both tumour imaging and therapy. Peptide ligands have been developed which show good binding specificity for ?v?6 and provide an opportunity to study the interaction in more detail by NMR. Such studies ideally require 15N and 13C labelled peptides, and recombinant expression within E. coli provides a cost effective way of generating isotopically labelled proteins and peptides. In this study we have used an insoluble fusion partner (ketosteroid isomerase) to produce high yields of recombinant peptide. The insoluble nature of the fusion allowed simple product recovery by cell lysis and centrifugation, and thorough washing of the insoluble pellet to remove contaminating proteins avoided the need for nickel-affinity chromatography in denaturing conditions which is the standard procedure. The protocol described here is convenient to scale-up and requires only one chromatography step (reverse-phase HPLC) which is comparable to solid-phase synthesis.
  • Wagstaff, J. et al. (2010). Two-dimensional heteronuclear saturation transfer difference NMR reveals detailed integrin alphavbeta6 protein-peptide interactions. Chemical Communications [Online] 46:7533-7535. Available at: http://dx.doi.org/10.1039/c0cc01846e.
    We report the first example of peptide-protein heteronuclear two-dimensional (2D) saturation transfer difference nuclear magnetic resonance (STD NMR). This method, resulting in dramatically reduced overlap, was applied to the interaction of the integrin alphavbeta6 with a known peptide ligand and highlights novel contact points between the substrate and target protein.
  • Williamson, R. (2010). Refolding of TIMP-2 from Escherichia coli Inclusion Bodies. Methods in Molecular Biology [Online] 622:111-121. Available at: http://dx.doi.org/10.1007/978-1-60327-299-5_7.
    The TIMP proteins contain six intramolecular disulfide bonds and form unfolded insoluble aggregates when expressed in E. coli. Eukaryotic expression systems provide the necessary post-translational modification apparatus to produce authentic TIMP but are comparatively slow and more expensive. This chapter describes the production of native TIMP-2 (both full-length and the N-terminal domain) from E. coli by in vitro refolding. The technique allows high-level intracellular expression and efficient isolation of the recombinant product without the use of fusion tags or partners. Protein purity after ion exchange and gel filtration chromatography was judged to be greater than 95% with yields of 15 mg/L from LB medium and 10 mg/L from minimal medium.
  • Wagstaff, J., Howard, M. and Williamson, R. (2010). Production of recombinant isotopically labelled peptide by fusion to an insoluble partner protein: generation of integrin alphavbeta6 binding peptides for NMR. Molecular BioSystems [Online] 6:2380-5. Available at: http://dx.doi.org/10.1039/c0mb00105h.
    The integrin alphavbeta6 is up-regulated in several cancers and has clinical potential for both tumour imaging and therapy. Peptide ligands have been developed which show good binding specificity for alphavbeta6 and provide an opportunity to study the interaction in more detail by NMR. Such studies ideally require (15)N and (13)C labelled peptides, and recombinant expression within E. coli provides a cost effective way of generating isotopically labelled proteins and peptides. In this study we have used an insoluble fusion partner (ketosteroid isomerase) to produce high yields of recombinant peptide. The insoluble nature of the fusion allowed simple product recovery by cell lysis and centrifugation, and thorough washing of the insoluble pellet to remove contaminating proteins avoided the need for nickel-affinity chromatography in denaturing conditions which is the standard procedure. The protocol described here is convenient to scale-up and requires only one chromatography step (reverse-phase HPLC) which is comparable to solid-phase synthesis.
  • Byrne, L. et al. (2009). Mapping of the ligand-binding site on the b' domain of human PDI: interaction with peptide ligands and the x-linker region. Biochemical Journal [Online] 423:209-217. Available at: http://dx.doi.org/10.1042/BJ20090565.
    PDI (protein disulfide-isomerase) catalyses the formation of native disulfide bonds of secretory proteins in the endoplasmic reticulum. PDI consists of four thioredoxin-like domains, of which two contain redox-active catalytic sites (a and a'), and two do not (b and b'). The b' domain is primarily responsible for substrate binding, although the nature and specificity of the substrate-binding site is still poorly understood. In the present study, we show that the b' domain of human PDI is in conformational exchange, but that its structure is stabilized by the addition of peptide ligands or by binding the x-linker region. The location of the ligand-binding site in b' was mapped by NMR chemical shift perturbation and found to consist primarily of residues from the core beta-sheet and alpha-helices 1 and 3. This site is where the x-linker region binds in the X-ray structure of b'x and we show that peptide ligands can compete with x binding at this site. The finding that x binds in the principal ligand-binding site of b' further supports the hypothesis that x functions to gate access to this site and so modulates PDI activity.
  • Wallis, A. et al. (2009). The ligand-binding b' domain of human protein disulphide-isomerase mediates homodimerization. Protein Science [Online] 18:2569-2577. Available at: http://dx.doi.org/10.1002/pro.270.
    Purified preparations of the recombinant b'x domain fragment of human protein-disulphide isomerase (PDI), which are homogeneous by mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis, comprise more than one species when analyzed by ion-exchange chromatography and nondenaturing polyacrylamide gel electrophoresis. These species were resolved and shown to be monomer and dimer by analytical ultracentrifugation and analytical size-exclusion chromatography. Spectroscopic properties indicate that the monomeric species corresponds to the "capped" conformation observed in the x-ray structure of the I272A mutant of b'x (Nguyen, Wallis, Howard, Haapalainen, Salo, Saaranen, Sidhu, Wierenga, Freedman, Ruddock, and Williamson, J Mol Biol 2008;383:1144-1155) in which the x region binds to a hydrophobic patch on the surface of the b' domain; conversely, the dimeric species has an "open" or "uncapped" conformation in which the x region does not bind to this surface. The larger bb'x fragment of human PDI shows very similar behavior to b'x and can be resolved into a capped monomeric species and an uncapped dimer. Preparations of recombinant b' domain of human PDI and of the bb' domain pair are found exclusively as dimers. Full-length PDI is known to comprise a mixture of monomeric and dimeric species, whereas the isolated a, b, and a' domains of PDI are found exclusively as monomers. These results show that the b' domain of human PDI tends to form homodimers--both in isolation and in other contexts--and that this tendency is moderated by the adjacent x region, which can bind to a surface patch on the b' domain.
  • Rowe, M. et al. (2009). Solution structure and dynamics of ERp18, a small endoplasmic reticulum resident oxidoreductase. Biochemistry [Online] 48:4596-4606. Available at: http://dx.doi.org/10.1021/bi9003342.
    Here we report the solution structure of oxidized ERp18 as determined using NMR spectroscopy. ERp18 is the smallest member of the protein disulfide isomerase (PDI) family of proteins to contain a Cys-Xxx-Xxx-Cys active site motif. It is an 18 kDa endoplasmic reticulum resident protein with unknown function although sequence similarity to individual domains of the thiol-disulfide oxidoreductase PDI suggests ERp18 may have a similar structure and function. Like the catalytic domains of PDI, ERp18 adopts a thioredoxin fold with a thioredoxin-like active site located at the N-terminus of a long kinked helix that spans the length of the protein. Comparison of backbone chemical shifts for oxidized and reduced ERp18 shows the majority of residues possess the same backbone conformation in both states, with differences limited to the active site and regions in close proximity. S(2) order parameters from NMR backbone dynamics were found to be 0.81 for oxidized and 0.91 for reduced ERp18, and these observations, in combination with amide hydrogen exchange rates, imply a more rigid and compact backbone for the reduced structure. These observations support a putative role for ERp18 within the cell as an oxidase, introducing disulfide bonds to substrate proteins, providing structural confirmation of ERp18's role as a thiol-disulfide oxidoreductase.
  • Lightbody, K. et al. (2008). Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins. Journal of Biological Chemistry [Online] 283:17681-17690. Available at: http://dx.doi.org/10.1074/jbc.M800123200.
    The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.
  • Williamson, R. et al. (2008). Dynamic characterisation of the netrin-like domain of human type 1 procollagen C-proteinase enhancer and comparison to the N-terminal domain of tissue inhibitor of metalloproteinases (TIMP). Molecular Biosystems [Online] 4:417-425. Available at: http://dx.doi.org/10.1039/b717901d.
    The backbone mobility of the C-terminal domain of procollagen C-proteinase enhancer (NTR(PCOLCE1)), part of a connective tissue glycoprotein, was determined using (15)N NMR spectroscopy. NTR(PCOLCE1) has been shown to be a netrin-like domain and adopts an OB-fold such as that found in the N-terminal domain of tissue inhibitors of metalloproteinases-1 (N-TIMP-1), N-TIMP-2, the laminin-binding domain of agrin and the C-terminal domain of complement protein C5. NMR relaxation dynamics of NTR(PCOLCE1) highlight conformational flexibility in the N-terminus, strand A and the proximal CD loop. This region in N-TIMP is known to be essential for inhibitory activity against the matrix metalloproteinases and suggests that this region is of equal importance for NTR(PCOLCE1), although the specific functional activity of the NTR(PCOLCE1) domain is still unknown. Dynamics observed within the structural core of NTR(PCOLCE1) that are not observed in N-TIMP molecules suggest that although the two domains have a similar architecture, the NTR(PCOLCE1) domain will show different thermodynamic properties on binding and hence the target molecule could be somewhat different from that observed for the TIMPs. ModelFree order parameters show that NTR(PCOLCE1) has more flexibility than both N-TIMP-1 and N-TIMP-2.
  • Nguyen, V. et al. (2008). Alternative conformations of the x region of human protein disulphide-isomerase modulate exposure of the substrate binding b' domain. Journal of Molecular Biology [Online] 383:1144-1155. Available at: http://dx.doi.org/10.1016/j.jmb.2008.08.085.
    Protein disulphide isomerase (PDI) is a key multi-domain protein folding catalyst in the endoplasmic reticulum. The b' domain of PDI is essential for the non-covalent binding of incompletely folded protein substrates. Earlier, we defined the substrate binding site in the b' domain of human PDI by modelling and mutagenesis studies. Here, we show by fluorescence and NMR that recombinant human PDI b'x (comprising the b' domain and the subsequent x linker region) can assume at least two different conformations in solution. We have screened mutants in the b'x region to identify mutations that favour one of these conformers in recombinant b'x, and isolated and characterised examples of both types. We have crystallised one mutant of b'x (I272A mutation) in which one conformer is stabilized, and determined its crystal structure to a resolution of 2.2 A. This structure shows that the b' domain has the typical thioredoxin fold and that the x region can interact with the b' domain by "capping" a hydrophobic site on the b' domain. This site is most likely the substrate binding site and hence such capping will inhibit substrate binding. All of the mutations we previously reported to inhibit substrate binding shift the equilibrium towards the capped conformer. Hence, these mutations act by altering the natural equilibrium and decreasing the accessibility of the substrate binding site. Furthermore, we have confirmed that the corresponding structural transition occurs in the wild type full-length PDI. A cross-comparison of our data with that for other PDI-family members, Pdi1p and ERp44, suggests that the x region of PDI can adopt alternative conformations during the functional cycle of PDI action and that these are linked to the ability of PDI to interact with folding substrates.
  • Povey, J. et al. (2008). The effect of peptide glycation on protein secondary structure. Journal of Structural Biology [Online] 161:151-161. Available at: http://dx.doi.org/10.1016/j.jsb.2007.10.004.
    Protein glycation is a non-enzymatic reaction between reducing sugars and amino groups that occurs in vivo and has been implicated in a number of disease states and pathologies including Alzheimer's and diabetes. Although glycation is thought to alter protein structure and function, there is currently little information on the structural consequences of this modification. We have used a model alpha-helix and a model beta-hairpin peptide, and NMR analysis, to investigate the effects of glycation upon secondary structure. Glycation of the dilysine motif within the alpha-helix peptide occurred preferentially at one lysine residue and resulted in severe disruption to the local secondary structure. The area immediately around the site of modification was extremely flexible and the peptide did not adopt a preferred conformation in this area of the helix in 30% TFE. Significant glycation of the beta-hairpin peptide was not detected and the structure was unchanged. These results show that glycation results in local secondary structure distortion of alpha-helices and that preferential glycation occurs in a sequence specific manner. The findings will allow us to interrogate the local environment in other peptides/proteins to predict the likelihood of glycation, and to model the potential effects such modification might have upon structure/function.
  • Alanen, H. et al. (2006). ERp27, a new non-catalytic endoplasmic reticulum-located human protein disulfide isomerase family member, interacts with ERp57. Journal of Biological Chemistry [Online] 281:33727-33738. Available at: http://dx.doi.org/10.1074/jbc.M604314200.
    Protein folding and quality control in the endoplasmic reticulum are critical processes for which our current understanding is far from complete. Here we describe the functional characterization of a new human 27.7-kDa protein (ERp27). We show that ERp27 is a two-domain protein located in the endoplasmic reticulum that is homologous to the non-catalytic b and b' domains of protein disulfide isomerase. ERp27 was shown to bind Delta-somatostatin, the standard test peptide for protein disulfide isomerase-substrate binding, and this ability was localized to the second domain of ERp27. An alignment of human ERp27 and human protein disulfide isomerase allowed for the putative identification of the peptide binding site of ERp27 indicating conservation of the location of the primary substrate binding site within the protein disulfide isomerase family. NMR studies revealed a significant conformational change in the b'-like domain of ERp27 upon substrate binding, which was not just localized to the substrate binding site. In addition, we report that ERp27 is bound by ERp57 both in vitro and in vivo by a similar mechanism by which ERp57 binds calreticulin.
  • Rapti, M. et al. (2006). Characterization of the AB loop region of TIMP-2. Involvement in pro-MMP-2 activation. Journal of Biological Chemistry [Online] 281:23386-23394. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16777853.
    Tissue inhibitor of metalloproteinases-2 (TIMP-2) is unique as it is the only member of the TIMP family that is involved in the cellular activation of promatrix metalloproteinase-2 (pro-MMP-2) by virtue of forming a trimolecular complex with membrane type 1 matrix metalloproteinase (MT1-MMP) on the cell surface. TIMP-4 is similar in structure to TIMP-2 but is unable to support the activation of the proenzyme. Several reports have highlighted the importance of the TIMP-2 C-terminal domain in the pro-MMP-2 activation complex; however, very little is known about the role of the extended AB loop of TIMP-2 in this mechanism even though it has been shown to interact with MT1-MMP. In this study we show by mutagenesis and kinetic analysis that it is possible to transfer the MT1-MMP binding affinity of the TIMP-2 AB loop to TIMP-4 but that its transplantation into TIMP-4 does not endow the inhibitor with pro-MMP-2 activating activity. However, transfer of both the AB loop and C-terminal domain of TIMP-2 to TIMP-4 generates a mutant that can activate pro-MMP-2 and so demonstrates that both these regions of TIMP-2 are important for the activation process.
  • Renshaw, P. et al. (2005). Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO Journal [Online] 24:2491-2498. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15973432.
    The secreted Mycobacterium tuberculosis complex proteins CFP-10 and ESAT-6 have recently been shown to play an essential role in tuberculosis pathogenesis. We have determined the solution structure of the tight, 1:1 complex formed by CFP-10 and ESAT-6, and employed fluorescence microscopy to demonstrate specific binding of the complex to the surface of macrophage and monocyte cells. A striking feature of the complex is the long flexible arm formed by the C-terminus of CFP-10, which was found to be essential for binding to the surface of cells. The surface features of the CFP-10.ESAT-6 complex, together with observed binding to specific host cells, strongly suggest a key signalling role for the complex, in which binding to cell surface receptors leads to modulation of host cell behaviour to the advantage of the pathogen.
  • Pirneskoski, A. et al. (2004). Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase. Journal of Biological Chemistry [Online] 279:10374-10381. Available at: http://dx.doi.org/10.1074/jbc.M312193200.
    Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.
  • Lightbody, K. et al. (2004). Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility. FEMS Microbiol Lett [Online] 238:255-262. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15336430.
    We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.
  • Renshaw, P. et al. (2004). Sequence-specific assignment and secondary structure determination of the 195-residue complex formed by the Mycobacterium tuberculosis proteins CFP-10 and ESAT-6. Journal of Biomolecular NMR [Online] 30:225-226. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15557808.
  • Carr, M. et al. (2003). Solution structure of the Mycobacterium tuberculosis complex protein MPB70: from tuberculosis pathogenesis to inherited human corneal desease. Journal of Biological Chemistry [Online] 278:43736-43743. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12917404.
    The closely related mycobacteria responsible for tuberculosis produce an unusually high number of secreted proteins, many of which are clearly implicated in pathogenesis and protective immunity. Falling within this category are the closely related proteins MPB70 and MPB83. The structure of MPB70 reveals a complex and novel bacterial fold, which has clear structural homology to the two C-terminal FAS1 domains of the cell adhesion protein fasciclin I, whose structures were reported very recently. Assessment of the surface features of MPB70, the sequence divergence between MPB70 and MPB83, the conservation of residues across a group of FAS1 domains, and the locations of disease-inducing mutations in betaig-h3 strongly suggests that MPB70 and MPB83 contain two functional surfaces on opposite faces, which are probably involved in binding to host cell proteins. This analysis also suggests that these functional surfaces are retained in the FAS1 proteins associated with mediating interactions between cells and the extracellular matrix (fasciclin I, periostin, and betaig-h3) and furthermore that some of the human corneal disease-inducing substitutions identified in betaig-h3 will perturb interactions at these sites.
  • Murphy, G. et al. (2003). Role of TIMPs (tissue inhibitors of metalloproteinases) in pericellular proteolysis: the specificity is in the detail. Biochemical Society Symposia [Online] 70:65-80. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14587283.
    Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor alpha-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.
  • Jones, G. et al. (2003). Sequence-specific assignment of the B-Myb DNA-binding domain (B-MybR2R3) bound to a 16 base-pair DNA target site corresponding to a regulatory site from the tom-1 gene. Journal of Biomolecular NMR [Online] 26:375-376. Available at: http://dx.doi.org/10.1023/A:1024005124375.
  • Alanen, H. et al. (2003). Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member. Journal of Biological Chemistry [Online] 278:28912-28920. Available at: http://dx.doi.org/10.1074/jbc.M304598200.
    Native disulfide bond formation in the endoplasmic reticulum is a critical process in the maturation of many secreted and outer membrane proteins. Although a large number of proteins have been implicated in this process, it is clear that our current understanding is far from complete. Here we describe the functional characterization of a new 18-kDa protein (ERp18) related to protein-disulfide isomerase. We show that ERp18 is located in the endoplasmic reticulum and that it contains a single catalytic domain with an unusual CGAC active site motif and a probable insertion between beta3 and alpha3 of the thioredoxin fold. From circular dichroism and NMR measurements, ERp18 is well structured and undergoes only a minor conformational change upon dithioldisulfide exchange in the active site. Guanidinium chloride denaturation curves indicate that the reduced form of the protein is more stable than the oxidized form, suggesting that it is involved in disulfide bond formation. Furthermore, in vitro ERp18 possesses significant peptide thiol-disulfide oxidase activity, which is dependent on the presence of both active site cysteine residues. This activity differs from that of the human PDI family in that under standard assay conditions it is limited by substrate oxidation and not by enzyme reoxidation. A putative physiological role for Erp18 in native disulfide bond formation is discussed.
  • Renshaw, P. et al. (2002). Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6*CFP-10 complex. Implications for pathogenesis and virulence. Journal of Biological Chemistry [Online] 277:21598-21603. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11940590.
    The proteins ESAT-6 and CFP-10 have been shown to be secreted by Mycobacterium tuberculosis and Mycobacterium bovis cells, to be potent T-cell antigens, and to have a clear but as yet undefined role in tuberculosis pathogenesis. We have successfully overexpressed both ESAT-6 and CFP-10 in Escherichia coli and developed efficient purification schemes. Under in vivo-like conditions, a combination of fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy have shown that ESAT-6 contains up to 75% helical secondary structure, but little if any stable tertiary structure, and exists in a molten globule-like state. In contrast, CFP-10 was found to form an unstructured, random coil polypeptide. An exciting discovery was that ESAT-6 and CFP-10 form a tight, 1:1 complex, in which both proteins adopt a fully folded structure, with about two-thirds of the backbone in a regular helical conformation. This clearly suggests that ESAT-6 and CFP-10 are active as the complex and raises the interesting question of whether other ESAT-6/CFP-10 family proteins (22 paired genes in M. tuberculosis) also form tight, 1:1 complexes, and if so, is this limited to their genome partner, or is there scope for wider interactions within the protein family, which could provide greater functional flexibility?
  • Williamson, R. et al. (2001). Tyrosine 36 plays a critical role in the interaction of the AB loop of tissue inhibitor of metalloproteinases-2 with matrix metalloproteinase-14. Journal of Biological Chemistry [Online] 276:32966-32970. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11390386.
    The tissue inhibitor of metalloproteinases-2 (TIMP-2) is potentially an important inhibitor of all known matrix metalloproteinases (MMPs). However, it has been shown to undergo specific interactions with both MMP-2 (gelatinase A) and MMP-14 (MT1-MMP), and it has been proposed that these three proteins function as a cell surface-based activation cascade for matrix metalloproteinases and as a focus of proteolytic activity. In this study, we have carried out mutagenesis and kinetic analyses to examine the unique interactions between the AB loop of TIMP-2 and MMP-14. The results demonstrate that the major binding contribution of the AB loop is due solely to residue Tyr-36 at the tip of the hairpin. From this work, we propose that TIMP-2 may be engineered to abrogate MMP-14 binding, whereas its binding properties for other MMPs, including MMP-2, are maintained. Mutants of TIMP-2 with more directed specificity may be of use in gene therapeutic approaches to human disease.
  • Kulasegaram, R. et al. (2001). In Vivo Evaluation of 111In-DTPA-N-TIMP-2 in Kaposi Sarcoma Associated with HIV Infection. European Journal of Nuclear Medicine [Online] 28:756-761. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11440037.
    Matrix metalloproteinases are the major agents responsible for the degradation of extracellular matrix and are produced at high levels by transformed and tumour cells, where they participate in the metastatic process by allowing local invasion. They are also more active at sites of new normal growth and angiogenesis. In the early stages of Kaposi sarcoma (KS), in vitro studies have demonstrated that vascular invasion can be inhibited by inhibitors of matrix metalloproteinases. Imaging of visceral and cutaneous KS presents a problem and therefore the potential use of a labelled inhibitor of metalloproteinases, N-TIMP-2, with indium-111 was thought to present a possible imaging tool. The biokinetics, dosimetry and potential for imaging with 111In-DTPA-N-TIMP-2 were assessed in five patients with HIV infection and KS. Between 103.1 and 108.0 MBq of this agent was injected into each patient, and the dynamic uptake over the kidneys was assessed, whole body scans were performed and blood samples were obtained. The clearance from the blood was rapid, with a first component half-time of 16.6+/-3.4 min and a second component half-time of 9.68+/-2.68 h. Two out of five patients experienced minor shivering but one of these patients was generally unwell before the study. The last three patients had no such problems. The tracer distributed predominantly to the kidneys and did not localise in other tissues. No KS lesions were clearly identified. 111In-DTPA-N-TIMP-2 can be successfully prepared and administered to patients safely, with a biodistribution and dosimetry which would allow its use as an imaging tracer. It is unlikely to be of use for imaging KS, but may have a role in other tumours that produce matrix metalloproteinases.
  • Williamson, R. (2001). Refolding of TIMP-2 from Escherichia Coli Inclusion Bodies. Methods in Molecular Biology [Online] 151:257-265. Available at: http://dx.doi.org/10.1385/1-59259-046-2:257.
    E. coli is a convenient host in which to express recombinant proteins. The technology is available to most laboratories as it is relatively inexpensive and does not require extensive expertise. The major drawback of E. coli as an expression host is the inability of the organism to carry out many posttrans-lational modifications, including glycosylation and disulphide bond formation. High-level intracellular expression of many mammalian proteins in E. coli results in the formation of large insoluble aggregates, known as inclusion bodies (1). These dense bodies consist predominantly of the misfolded recombinant product, together with components of the transcription/translation machinery (i.e., RNA polymerase, ribosomal RNA, and plasmid DNA). The TIMPs are invariably insoluble when expressed in E. coli, their folding requirement for the formation of 6 disulphide bonds being incompatible with the reducing environment of the E. coli cell. Fortunately, active, correctly folded recombinant protein can often be recovered from insoluble inclusion bodies by a process of solubilization and in vitro refolding (2–3). Indeed, inclusion body formation has the advantage that the recombinant product often accumulates to high levels in the cell (up to 30% of total cell protein) and allows easy isolation of that protein, a relatively pure and stable form (inclusion bodies are typically >50% recombinant protein).
  • Bloemink, M. et al. (2001). Sequence-specific assignment and determination of the secondary structure of the 163-residue M. tuberculosis and M. bovis antigenic protein mpb70. Journal of Biomolecular NMR [Online] 20:185-186. Available at: http://dx.doi.org/10.1023/A:1011239727839.
  • Giersing, B. et al. (2001). Synthesis and Characterization of 111In-DTPA-N-TIMP-2: A Radiopharmaceutical for Imaging Matrix Metalloproteinase Expression. Bioconjugate Chemistry [Online] 12:964-971. Available at: http://dx.doi.org/10.1021%2Fbc010028f.
    The matrix metalloproteinases (MMPs) are enzymes involved in the turnover of the extracellular matrix. Their overexpression in tumors is implicated in the metastatic process and may provide a target for diagnostic tumor imaging by using a radiolabeled inhibitor. MMPs are inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs). Thus, TIMPs are potential targeting molecules which could be used as vehicles for selective radionuclide delivery by virtue of their binding to MMPs. The aim of this work was to produce a radiopharmaceutical with which to evaluate this potential. The 127 amino acid N-terminal domain of recombinant human TIMP-2 (N-TIMP-2) was conjugated with the bifunctional chelator diethylenetriamine pentaacetic acid (DTPA). Singly modified DTPA-N-TIMP-2 conjugate (identified by electrospray ionization mass spectrometry) was isolated by anion-exchange chromatography. The primary site of DTPA modification on N-TIMP-2 was mapped to lysine-116, which is distant from the site of MMP interaction. The conjugate was radiolabeled with indium-111 to give 111In-DTPA-N-TIMP-2 with a specific activity of at least 4 MBq/microg and a radiochemical yield and purity of >95%, by incubation with 111InCl3, without need for postlabeling purification. The product was sterile, pyrogen-free, and stable in serum over 48 h and retained full inhibitory activity in a fluorimetric binding assay. With these attributes, 111In-DTPA-N-TIMP-2 is a suitable radiopharmaceutical for in vivo biological and clinical investigation of the potential benefits of imaging MMP expression.
  • Blower, P. et al. (2001). Synthesis and Characterization of 111In?DTPA?N-TIMP-2:? A Radiopharmaceutical for Imaging Matrix Metalloproteinase Expression. Bioconjugate Chemistry [Online] 12:964-971. Available at: http://dx.doi.org/10.1021/bc010028f.
    The matrix metalloproteinases (MMPs) are enzymes involved in the turnover of the extracellular matrix. Their overexpression in tumors is implicated in the metastatic process and may provide a target for diagnostic tumor imaging by using a radiolabeled inhibitor. MMPs are inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs). Thus, TIMPs are potential targeting molecules which could be used as vehicles for selective radionuclide delivery by virtue of their binding to MMPs. The aim of this work was to produce a radiopharmaceutical with which to evaluate this potential. The 127 amino acid N-terminal domain of recombinant human TIMP-2 (N-TIMP-2) was conjugated with the bifunctional chelator diethylenetriamine pentaacetic acid (DTPA). Singly modified DTPA?N-TIMP-2 conjugate (identified by electrospray ionization mass spectrometry) was isolated by anion-exchange chromatography. The primary site of DTPA modification on N-TIMP-2 was mapped to lysine-116, which is distant from the site of MMP interaction. The conjugate was radiolabeled with indium-111 to give 111In?DTPA?N-TIMP-2 with a specific activity of at least 4 MBq/?g and a radiochemical yield and purity of >95%, by incubation with 111InCl3, without need for postlabeling purification. The product was sterile, pyrogen-free, and stable in serum over 48 h and retained full inhibitory activity in a fluorimetric binding assay. With these attributes, 111In?DTPA?N-TIMP-2 is a suitable radiopharmaceutical for in vivo biological and clinical investigation of the potential benefits of imaging MMP expression.
  • Blower, P. et al. (2000). Matrix metalloproteinases as a target for tumor imaging: In vivo evaluation of radiolabeled N-TIMP-2. Journal of Nuclear Medicine 41:258P-258P.
  • Butler, G. et al. (1999). The specificity of TIMP-2 for matrix metalloproteinases can be modified by single amino acid mutations. Journal of Biological Chemistry 274:20391-20396.
    Residues 1-127 of human TIMP-8 (N-TIMP-2), comprising three of the disulfide-bonded loops of the TIMP-8 molecule, is a discrete protein domain that folds independently of the C-terminal domain. This domain has been shown to be necessary and sufficient for metalloproteinase inhibition and contains the major sites of interaction with the catalytic N-terminal domain of active matrix metalloproteinases (MMPs), Residues identified as being involved in the interaction with MMPs by NMR chemical shift perturbation studies and TIMP/MMP crystal structures have been altered by site directed mutagenesis. We show, by measurement of association rates and apparent inhibition constants, that the specificity of these N-TIMP-2 mutants for a range of MMPs can be altered by single site mutations in either the TIMP "ridge" (Cys(1)-Cys(3) and Ser(68)-Cys(72)) Or the flexible AB loop (Ser(31)-Ile(41)). This work demonstrates that it is possible to engineer TIMPs with altered specificity and suggests that this form of protein engineering may be useful in the treatment of diseases such as arthritis and cancer where the selective inhibition of key MMPs is desirable.
  • Williamson, R. et al. (1999). The effect of matrix metalloproteinase complex formation on the conformational mobility of tissue inhibitor of metalloproteinases-2 (TIMP-2). Journal of Biological Chemistry [Online] 274:37226-37232. Available at: http://dx.doi.org/10.1074/jbc.274.52.37226.
    The backbone mobility of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) was determined both for the free protein and when bound to the catalytic domain of matrix metalloproteinase-3 (N-MMP-3). Regions of the protein with internal motion were identified by comparison of the T(1) and T(2) relaxation times and (1)H-(15)N nuclear Overhauser effect values for the backbone amide (15)N signals for each residue in the sequence. This analysis revealed rapid internal motion on the picosecond to nanosecond time scale for several regions of free N-TIMP-2, including the extended beta-hairpin between beta-strands A and B, which forms part of the MMP binding site. Evidence of relatively slow motion indicative of exchange between two or more local conformations on a microsecond to millisecond time scale was also found in the free protein, including two other regions of the MMP binding site (the CD and EF loops). On formation of a tight N-TIMP-2. N-MMP-3 complex, the rapid internal motion of the AB beta-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB beta-hairpin is not a feature of all members of the TIMP family; therefore, the binding of this highly mobile region to a site distant from the catalytic cleft of the MMPs suggests a key role in TIMP-2 binding specificity.
  • Hutton, M. et al. (1999). Analysis of the interaction of TIMP-2 and MMPs: engineering the changes. Annals of the New York Academy of Sciences [Online] 878:524-527. Available at: http://dx.doi.org/10.1111/j.1749-6632.1999.tb07716.x.
  • Glaser, M. et al. (1998). Structural characterisation and bioconjugation of an active ester containing oxorhenium(V) complex incorporating a thioether donor. Journal of the Chemical Society-Dalton Transactions [Online]:3087-3092. Available at: http://dx.doi.org/10.1039/a804717k.
    A simple new 2,3,5,6-tetrafluorophenyl ester containing diamide-thioether-thiol bifunctional chelating agent LH3, HS(CH2)(2)SCH2C(O)NHCH2C(O)NH(CH2)(3)C(O)OC6HF4, has been synthesised. The key intermediates were prepared using standard peptide chemistry procedures. Reaction of LH3 with [Bu4N][ReOCl4] formed an uncharged oxorhenium(v) complex, which was characterised by X-ray structural analysis. The five-co-ordinate complex showed approximately square-pyramidal geometry with an apical oxo group and a basal ligand set comprising a deprotonated thiol group, two deprotonated amide groups, and a thioether group. A second complex of stoichiometry [ReO(LH2)(2)]Cl was formed by reaction of LH3 with a rhenium(v) gluconate intermediate in water at pH 4.7. The 1 : 1 complex [ReOL] was conjugated with the small protein N-TIMP-2 by aminolysis at a lysine residue, to form a 1: 1 adduct as established by electrospray mass spectrometry.
  • Muskett, F. et al. (1998). High resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 and characterization of its interaction site with matrix metalloproteinase-3. Journal of Biological Chemistry [Online] 273:21736-21743. Available at: http://dx.doi.org/10.1074/jbc.273.34.21736.
    The high resolution structure of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) in solution has been determined using multidimensional heteronuclear NMR spectroscopy, with the structural calculations based on an extensive set of constraints, including 3132 nuclear Overhauser effect-based distance constraints, 56 hydrogen bond constraints, and 220 torsion angle constraints (an average of 26.9 constraints/residue). The core of the protein consists of a five-stranded beta-barrel that is homologous to the beta-barrel found in the oligosaccharide/oligonucleotide binding protein fold. The binding site for the catalytic domain of matrix metalloproteinases-3 (N-MMP-3) on N-TIMP-2 has been mapped by determining the changes in chemical shifts on complex formation for signals from the protein backbone (15N, 13C, and 1H). This approach identified a discrete N-MMP-3 binding site on N-TIMP-2 composed of the N terminus of the protein and the loops between beta-strands AB, CD, and EF. The beta-hairpin formed from strands A and B in N-TIMP-2 is significantly longer than the equivalent structure in TIMP-1, allowing it to make more extensive binding interactions with the MMP catalytic domain. A detailed comparison of the N-TIMP-2 structure with that of TIMP-1 bound to N-MMP-3 (Gomis-Ruth, F.-X., Maskos, K., Betz, M., Bergner, A., Huber, R., Suzuki, K., Yoshida, N., Nagase, H., Brew, K., Bourne, G. P., Bartunik, H. & Bode, W. (1997) Nature 389, 77-80) revealed that the core beta-barrels are very similar in topology but that the loop connecting beta-strands CD (P67-C72) would need to undergo a large conformational change for TIMP-2 to bind in a similar manner to TIMP-1.
  • Williamson, R. et al. (1997). Mapping the binding site for matrix metalloproteinase on the N-terminal domain of the tissue inhibitor of metalloproteinases-2 by NMR chemical shift perturbation. Biochemistry [Online] 36:13882-13889. Available at: http://dx.doi.org/10.1021/bi9712091.
    Changes in the NMR chemical shift of backbone amide nuclei (H-1 and N-15) have been used to map the matrix metalloproteinase (MMP) binding site on the N-terminal domain of the tissue inhibitor of metalloproteinase-2 (N-TIMP-2). Amide chemical shift changes were measured on formation of a stable complex with the catalytic domain of stromelysin-l (N-MMP-3). Residues with significantly shifted amide signals mapped specifically to a broad site covering one face of the molecule. This site (the MMP binding site) consists primarily of residues 1-11, 27-41, 68-73, 87-90, and 97-104. The site overlaps with the OB-fold binding site seen in other proteins that share the same five-stranded beta-barrel topology. Sequence conservation data and recent site-directed mutagenesis studies are discussed in relation to the MMP binding site identified in this work.

Datasets / databases

  • Rowe, M. et al. (2009). 1H, 13C, 15N chemical shift assignments for reduced ERp18. [NMR-STAR formatted text file]. Available at: http://www.bmrb.wisc.edu/data_library/generate_summary.php?bmrbId=7430.
    Citation: Rowe, Michelle, L.; Ruddock, Lloyd, W.; Kelly, Geoff; Schmidt, Jürgen, M.; Williamson, Richard, A.; Howard, Mark, J.; "Solution Structure and Dynamics of ERp18: a Small ER Resident Oxidoreductase," Biochemistry 48(21), 4596-4606 (2009). PubMed: 19361226. DOI: 10.1021/bi9003342. System Studied: ERp18. System Components: ERp18, Polymer, 157 residues, 17771 Da. Molecular Description: Classification: Oxidoreductase, Structure Weight: 17801.10 Da, Molecule: Thioredoxin domain-containing protein 12, Polymer: 1, Type: polypeptide(L), Length: 157, Chains: A, EC#: 1.8.4.2, Fragment: UNP residues 24-172. Natural Source: Common Name: Human. Taxonomy ID: 9606. Superkingdom: Eukaryota. Kingdom: Metazoa. Genus/species: Homo sapiens. Source: Scientific Name: Homo sapiens, Common Name: Human, Expression System: Escherichia coli. Experimental Data: NMR parameters. Type: Assigned Chemical Shifts. Number of Values: 601. Sets: 1.
  • Rowe, M. et al. (2009). Solution structure of Oxidised ERp18. [PDB formatted text file]. Available at: http://www.rcsb.org/pdb/explore/explore.do?pdbId=2K8V.
    Primary Citation: Rowe, M.L.; Ruddock, L.W.; Kelly, G.; Schmidt, J.M.; Williamson, R.A.; Howard, M.J. "Solution structure and dynamics of ERp18, a small endoplasmic reticulum resident oxidoreductase." Journal: (2009) Biochemistry 48: 4596-4606. PubMed: 19361226. DOI: 10.1021/bi9003342.
    Molecular Description: Classification: Oxidoreductase, Structure Weight: 17801.10 Da, Molecule: Thioredoxin domain-containing protein 12, Polymer: 1, Type: polypeptide(L), Length: 157, Chains: A, EC#: 1.8.4.2, Fragment: UNP residues 24-172. Source: Scientific Name: Homo sapiens, Common Name: Human, Expression System: Escherichia coli.
  • Rowe, M. et al. (2009). 1H, 13C, 15N chemical shift assignments for oxidised ERp18. [NMR-STAR formatted text file]. Available at: http://www.bmrb.wisc.edu/data_library/generate_summary.php?bmrbId=15964.
    Citation: Rowe, Michelle, L.; Ruddock, Lloyd, W.; Kelly, Geoff; Schmidt, Jürgen, M.; Williamson, Richard, A.; Howard, Mark, J.; "Solution Structure and Dynamics of ERp18: a Small ER Resident Oxidoreductase," Biochemistry 48(21), 4596-4606 (2009). PubMed: 19361226. DOI: 10.1021/bi9003342. System Studied: ERp18. System Components: ERp18, Polymer, 157 residues, 17771 Da. Molecular Description: Classification: Oxidoreductase, Structure Weight: 17801.10 Da, Molecule: Thioredoxin domain-containing protein 12, Polymer: 1, Type: polypeptide(L), Length: 157, Chains: A, EC#: 1.8.4.2, Fragment: UNP residues 24-172. Natural Source: Common Name: Human. Taxonomy ID: 9606. Superkingdom: Eukaryota. Kingdom: Metazoa. Genus/species: Homo sapiens. Source: Scientific Name: Homo sapiens, Common Name: Human, Expression System: Escherichia coli. Experimental Data: NMR parameters. Type: Assigned Chemical Shifts. Number of Values: 1760. Sets: 1.
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