The Effect of Matrix Metalloproteinase Complex Formation on the Conformational Mobility of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2)
1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês
10.1074/jbc.274.52.37226
ISSN1083-351X
AutoresRichard A. Williamson, Frederick W. Muskett, Mark J. Howard, Robert B. Freedman, Mark D. Carr,
Tópico(s)Peptidase Inhibition and Analysis
ResumoThe 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 1and T 2 relaxation times and1H-15N nuclear Overhauser effect values for the backbone amide 15N 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 β-hairpin between β-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 β-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB β-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. 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 1and T 2 relaxation times and1H-15N nuclear Overhauser effect values for the backbone amide 15N 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 β-hairpin between β-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 β-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB β-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. matrix metalloproteinase catalytic domain of human MMP-3 (stromelysin-1) nuclear Overhauser effect tissue inhibitor of metalloproteinases N-terminal domain (residues 1–127) of human TIMP-2 root-mean-square deviation Breakdown of the extracellular matrix is an important event in many normal and pathological processes, such as growth, wound repair, tumor metastasis, and arthritis (1Birkedal-Hanson H. Curr. Opin. Cell Biol. 1995; 7: 728-735Crossref PubMed Scopus (980) Google Scholar, 2Cawston T.E. Mol. Med. Today. 1998; 4: 130-137Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 3Yong V.W. Krekowski C.A. Forsyth P.A. Bell R. Edwards D.R. Trends Neurosci. 1998; 21: 75-80Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). A large family of zinc-dependent proteinases, the matrix metalloproteinases (MMPs),1 are thought to be primarily responsible for this matrix catabolism. The activities of the MMP family in the extracellular matrix are highly regulated by transcriptional control, zymogen activation, and inhibition by a family of specific protein inhibitors, the tissue inhibitors of metalloproteinases or TIMPs (4Gormez D.E. Alonso D.F. Yoshiji H. Thoreirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar). The TIMPs bind tightly to the active proteinases to form an inactive TIMP·MMP complex (5Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (246) Google Scholar). Four mammalian TIMP proteins have now been identified (TIMP-1 to -4) (6Docherty A.J.P. Lyons A. Smith B.J. Wright E.M. Stephens P.E. Harris T.J.R. Murphy G. Reynolds J.J. Nature. 1985; 318: 66-69Crossref PubMed Scopus (601) Google Scholar, 7Boone T.C. Johnson M.J. DeClerck Y.A. Langley K.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2800-2804Crossref PubMed Scopus (186) Google Scholar, 8Apte S.S. Mattei M.-G. Olsen B.R Genomics. 1994; 19: 86-90Crossref PubMed Scopus (201) Google Scholar, 9Greene K. Wang M. Liu Y. Raymond L.A. Rosen C. Shi Y.E J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar), and their high degree of sequence similarity and conservation of 12 Cys residues suggests that each consists of the same basic fold but with some variations in loop structures and glycosylation. The location of the MMP inhibitory site on the TIMP molecule has been shown to reside predominantly in the N-terminal two-thirds of the protein, defined by the first three disulfide bonds. This domain (N-TIMP) can be expressed independently to generate a fully folded, stable, and active inhibitor (10Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (292) Google Scholar, 11Williamson R.A. Bartels H. Murphy G. Freedman R.B. Protein Eng. 1994; 7: 1035-1040Crossref PubMed Scopus (18) Google Scholar). High resolution three-dimensional structures are now available for both the active N-terminal domain of TIMP-2 (12Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and for the full-length inhibitor (13Tuuttila A. Morgunova E. Bergmann U. Lindqvist Y. Maskos K. Fernandez-Catalan C. Bode W. Tryggvason K. Scheider G. J. Mol. Biol. 1998; 284: 1133-1140Crossref PubMed Scopus (84) Google Scholar). Crystal structures have also been published for full-length TIMP-1 and TIMP-2 in complexes with the catalytic domains of MMP-3 (N-MMP-3) and MT1-MMP, respectively (14Gomis-Ruth F.X. Maskos K. Bergner A. Huber R. Suzuki K. Yoshida N. Nagase H. Brew K. Bourenkov G.P. Bertunik H. Bode W. Nature. 1997; 389: 77-81Crossref PubMed Scopus (520) Google Scholar, 15Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (317) Google Scholar). The structures of the TIMP·MMP complexes, together with NMR data on chemical shift perturbation seen for N-TIMP-2 on complex formation with N-MMP-3 (16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar), have identified the key features of the TIMP inhibitory binding site. It is now clear that the N terminus of TIMP (residues 1–5), together with the two loops with which it is disulfide-bonded, form a “wedge”-like structure that interacts with the active site cleft of the proteinase. A further region in the N-terminal domain of TIMP that interacts with the proteinase is the loop between β-strands A and B. In TIMP-2, this region is extended by 7 residues compared with TIMP-1 and is therefore capable of making more extensive interactions with the proteinase. The involvement of the extended AB β-hairpin in TIMP-2/MMP interactions was first proposed on the basis of chemical shift changes observed on binding of N-TIMP-2 to N-MMP-3 (16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar), and further confirmed in the crystal structure for the TIMP-2·MT1-MMP complex (15Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (317) Google Scholar). NMR studies of N-TIMP-2 provided the first three-dimensional structure for the inhibitor and allowed it to be identified as a member of the OB (oligonucleotide/oligosaccharide)-fold protein family (17Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J.P. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745-11759Crossref PubMed Scopus (95) Google Scholar). This work was later extended to provide a high resolution structure of N-TIMP-2 using heteronuclear NMR-based methods (12Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These studies provided some insights into the dynamics of N-TIMP-2, particularly for the AB β-hairpin, which we proposed to be a highly flexible structure due to the relatively narrow line widths seen for signals in this region and the lack of long range NOEs. Furthermore, we suggested that another region of the N-TIMP-2 binding site (Ser68–Cys72) was in relatively slow exchange between multiple conformations interconverting on a millisecond time scale, as the backbone amide resonances for these residues were missing from NMR spectra (16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar). To provide a more detailed picture of the backbone mobility of N-TIMP-2 and to assess how MMP complex formation affects these motions, the values of T 1, T 2, and heteronuclear 1H-15N NOE have now been determined for the backbone amide 15N signals of N-TIMP-2 when free in solution and when bound to N-MMP-3. These studies show that several regions of N-TIMP-2 have significant local mobility on time scales both slower or faster than the overall tumbling time of the protein (estimated to be 10 ns). In particular, rapid motion was seen for the AB β-hairpin of free N-TIMP-2, and this mobility was lost on complex formation with N-MMP-3, suggesting that the extended AB loop is a general and important feature of the TIMP-2 binding site. 15N-Labeled human N-TIMP-2 was expressed in Escherichia coli and refolded from intracellular inclusion bodies as described previously (16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar, 18Williamson R.A. Natalia D. Gee C.K. Murphy G. Carr M.D. Freedman R.B. Eur. J. Biochem. 1996; 241: 476-483Crossref PubMed Scopus (34) Google Scholar). The expression vector for N-MMP-3 was kindly provided by A. Marcy (Merck), and the recombinant protein expressed and purified as described by Marcy et al. (19Marcy A.I. Eiberger L.L. Harrison R. Chan H.K. Hutchinson N.I. Hagmann W.K. Cameron P.M. Boulton D.A. Hermes J.D. Biochemistry. 1991; 30: 6476-6483Crossref PubMed Scopus (97) Google Scholar). N-MMP-3 was activated byp-aminophenylmercuric acetate and incubated with15N-labeled N-TIMP-2 to produce the N-TIMP-2·N-MMP-3 complex (16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar). The complex was purified from any free N-TIMP-2 or N-MMP-3 by gel filtration chromatography on Sephacryl S-100 (16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar). NMR experiments were carried out on 0.3-ml samples of 1.5 mm15N-labeled N-TIMP-2 (25 mm sodium phosphate buffer, 100 mm NaCl, pH 6.5) or 0.5 mm15N-labeled N-TIMP-2·N-MMP-3 complex (5 mmdeuterated-imidazole/HCl, 100 mm NaCl, 5 mmCaCl2, pH 6.5) in 5-mm Shigemi tubes. D2O was added to the NMR samples to a final concentration of 10% (v/v). The NMR experiments were carried out at 35 °C on a Varian INOVA spectrometer operating at 1H frequency of 600 MHz. The spectra used to determineT 1, T 2, and1H-15N NOE values for the backbone amide15N signals of both free and N-MMP-3 bound N-TIMP-2 were recorded with acquisition times of 16.8 ms in F1 and 142 or 148 ms in F2 using the sensitivity enhanced pulse sequences described by Farrow et al. (20Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay F.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2030) Google Scholar). The 1H carrier was centered at the water frequency for the T 1and T 2 measurements and at the center of the amide region for the heteronuclear 1H-15N NOE experiments. The 15N carrier was placed in the center of the amide region. In the case of free N-TIMP-2, spectra used to determineT 1 values were acquired with 16 transients per increment and a relaxation delay of 4.4 s,T 2 values with 32 transients per increment and a relaxation delay of 3 s, and 1H-15N NOEs with 128 transients per increment and a relaxation delay of 8 s. The T 1 values were calculated from a series of spectra recorded with T 1 relaxation delays of 10.8, 64.9, 259.9 (×2), 389.8, 563.1 (×2), 866.2, and 1234.4 ms. Similarly, to determine T 2 values for free N-TIMP-2 spectra were acquired with T 2relaxation delays of 15.7, 31.5 (×2), 47.2, 62.9, 94.4 (×2), 110.1, and 141.6 ms. The magnitude of the 1H-15N NOE for the backbone amide 15N signals was calculated from a pair of spectra recorded either with or without presaturation of backbone amide 1H resonances during the relaxation delay. For N-TIMP-2 bound to N-MMP-3, spectra used to determine backbone amideT 1 values were recorded with 56 transients per increment and a relaxation delay of 5 s, T 2values with 80 transients per increment and a relaxation delay of 3 s, and 1H-15N NOEs with 128 increments per transient and a relaxation delay of 8 s. TheT 1 values were calculated from a series of spectra acquired with T 1 relaxation delays of 11.1, 66.3, 254.2 (×2), 375.8, 552.7 (×2), 862.2, and 1238.0 ms. In the case of T 2, values were determined from spectra recorded with T 2 relaxation delays of 15.7, 31.5 (×2), 47.2, 62.9, 94.4 (×2), 110.2, and 141.7 ms. The size of the heteronuclear 1H-15N NOE for the backbone amide 15N resonances of N-TIMP-2 bound to N-MMP-3 was determined as described for the free protein. The NMR spectra were processed on a Silicon Graphics Indigo 2 workstation using the program NMRPipe (21Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11837) Google Scholar). To increase the resolution in the final spectra the number of data points in F1 was extended 2-fold by linear prediction, and both F1 and F2 were zero-filled once. The spectra were examined and peak heights determined using the program XEASY (22Bartels C. Xia T.H. Billeter M. Guntert P. Wuthrich K. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1614) Google Scholar). The T 1 and T 2 values for backbone amide 15N signals were calculated by non-linear, least-squares fitting of the observed changes in peak heights to appropriate single exponential functions (23Palmer III, A.G. Rance M. Wright P.E. J. Am. Chem. Soc. 1991; 113: 4371-4378Crossref Scopus (599) Google Scholar, 24Stone M.J. Fairbrother W.J. Palmer III, A.G. Reizer J. Saier Jr., M.H. Wright P.E. Biochemistry. 1992; 31: 4394-4406Crossref PubMed Scopus (236) Google Scholar), using the program SigmaPlot. The values of steady-state 1H-15N NOEs were determined from the peak heights measured in spectra recorded either with (I s) or without (I o) presaturation of backbone amide1H resonances during the relaxation delay, according to the formula NOE = (I s −I o)/I o (25Noggle J.H. Schirmer R.E. The Nuclear Overhauser Effect: Chemical Applications. Academic Press, New York1971Google Scholar). This study provides a detailed picture of the backbone dynamics of the inhibitory domain of TIMP-2 (N-TIMP-2), and identifies those regions of the protein that undergo substantial changes in backbone mobility on formation of a stable complex with the MMP-3 catalytic domain (N-MMP-3).T 1, T 2, and heteronuclear1H-15N NOE measurements were obtained for the majority of the backbone amide 15N signals of free N-TIMP-2 (102/121) and for N-TIMP-2 in the complex (98/121). The residues for which no relaxation data could be obtained were Cys1–Cys3, Val6, Cys13, Asp16, Ser68–Val71, Asp77, Lys81, Glu83, Phe103, Thr113, Ser117, His120, Arg121, and Met124 in free N-TIMP-2, and Cys1–Val6, Asn14, Ile35, Lys40, Ser68–Gly73, Ser75, Lys81, Lys89, Asp93, Cys101, Thr112, Thr113, Tyr120, and Arg121 in the complex (residues 5, 8, 56, 39, 67, and 106 are proline). Backbone amide signals have not previously been detected for residues Cys1–Ser2 and Ser68–Ala70 in free N-TIMP-2 and for Cys1–Ser4, Asn14, Ile35, Ile40, Ser68–Ala70, Cys72, Ser75, The112, Thr113, His120, and Arg121 in N-TIMP-2 bound to N-MMP-3 (12Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar). 15N relaxation data for the remaining residues could not be determined due to peak overlap in the spectra or very broad signals that prevented accurate measurement of peak heights. For residues Lys27, Val71, and Thr112of free N-TIMP-2, and Tyr36, Gln49, Glu83, and Tyr122 of N-TIMP-2 in the complex, it was possible to determine the size of the1H-15N NOE but reliable estimates ofT 1 and T 2 could not be obtained. The T 2 values for backbone amide 15N signals of proteins are sensitive to both fast and slow local motions of the polypeptide backbone. NMR signals with T 2values significantly longer than the mean are indicative of rapid local motion on a time scale (picosecond to nanosecond) significantly shorter than the overall tumbling time (τ m) of the protein, and have relatively narrow line widths. In contrast, signals with T 2 values significantly shorter than the mean have relatively broad line widths and arise from exchange between two or more states (conformations) with different local environments. To significantly broaden NMR signals and decreaseT 2 values, this exchange must be on a time scale (microsecond to millisecond) similar to the chemical shift difference between the two states (so-called intermediate exchange). If the chemical shift difference between the two states is considerable (>100 Hz), then intermediate exchange will broaden the NMR signals to such an extent that they can no longer be detected. Conformational exchange on time scales significantly longer that the chemical shift difference will result in the different states being detected as separate signals, while conformational exchange on time scales significantly shorter than the chemical shift difference will result in sharp signals at an intermediate frequency. The size of the 1H-15N NOE for backbone amide signals is also sensitive to local mobility of the peptide chain on a picosecond to nanosecond time scale (i.e. significantly faster than the overall tumbling rate of the protein). Regions of the protein backbone with rapid local motion are characterized by large negative 1H-15N NOEs. The T 2 relaxation times for the backbone amide 15N signals of free N-TIMP-2 are shown in Fig. 1 A. The mean and standard deviation (indicated on the chart) were calculated after omitting the data for 3 residues which were judged to have unusually long T 2 values (Gly32, Gly92, and Glu127). Fig. 1 A clearly shows five regions where the T 2 values are significantly longer than the mean. The N terminus (residues Ser4, His7, and Gln10), the end of β-strand A, the AB loop and the beginning of strand B (the AB β-hairpin, residues Ser31–Ile35, Gly37, and Lys41), the region between the Cys1–Cys72 disulfide bond and strand D (residues Asp77, Gly79–Gly80), the loop between strands D and E (residues Gly92–Asp93) and the C terminus of the protein (residues Gly125–Glu127). Residue Lys58 (in the loop between strands B and C) is also raised. These elevated T 2 values suggest that these residues experience rapid internal motion on a picosecond to nanosecond time scale. The 1H-15N NOE results (Fig. 1 B) identified very similar regions of N-TIMP-2 as having rapid local mobility. Large negative NOEs were recorded for residues near the N and C termini (Ser4, His7, and Gly125–Glu127), in the AB β-hairpin (Ser31–Lys41), and in the loops between strands B and C (Lys58), the Cys1–Cys72 disulfide bond and strand D (Asp77 and Lys82) and strands D and E (Gly92–Asp93). The T 2and 1H-15N NOE data for free N-TIMP-2 are summarized on the loop diagram in Fig. 2 A.FIG. 2Loop diagram of N-TIMP-2 showing secondary structure elements and the backbone mobility for each residue in free N-TIMP-2 (A) and N-TIMP-2 bound to N-MMP-3 (B). Residues are colored according to the following scheme: red (rapid picosecond to nanosecond internal motion), heteronuclear 1H-15N NOE < −0.34; green (slow microsecond to millisecond internal motion), T 2 values < mean − S.D.;blue (no significant internal motion)1H-15N NOE > −0.34 andT 2 > mean + S.D.; white (no data), residues for which data could not be collected for relaxation analysis;gray (slow microsecond to millisecond internal motion), residues in the CD loop judged to be in intermediate conformational exchange due to very broad (Val71 and Cys72) and missing (Ser68–Ala70) backbone amide signals (12Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 16Williamson R.A. Carr M.D. Frenkiel T.A. Feeney J. Freedman R.B. Biochemistry. 1997; 36: 13882-13889Crossref PubMed Scopus (113) Google Scholar).View Large Image Figure ViewerDownload (PPT) The T 2 data for free N-TIMP-2 also identified several regions of the molecule with significantly shorterT 2 relaxation times than the mean, suggesting exchange between two or more conformations on the microsecond to millisecond time scale (Fig. 1 A). Residues Gln49–Lys51 near the end of strand B, residue Ile60 in the loop between strands B and C, and residues His97–Cys101 in strand E and the EF loop all have T 2 values below a threshold of 1 standard deviation from the mean (Figs. 1 A and 2 A). The T 1 values for the backbone amide15N signals of free N-TIMP-2 showed very little variation with sequence (data not shown). The average T 1was found to be 0.807 with a standard deviation of 0.088. TheT 1/T 2 ratios for N-TIMP-2 were used to estimate the overall rotational correlation time (τ m) of the molecule (26Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1829) Google Scholar). A value of 10 ns (at 35 °C) was obtained, which is comparable to the τ m values reported for several other proteins on the basis of T 1 and T 2data (26Kay L.E. Torchia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1829) Google Scholar, 27Broadhurst R.W. Hardman C.H. Thomas J.O. Laue E.D. Biochemistry. 1995; 34: 16608-16617Crossref PubMed Scopus (45) Google Scholar, 28Polshakov V.I. Williams M.A. Gargaro A.R. Frenkiel T.A. Westley B.R. Chadwick M.P. May F.E.B. Feeney J. J. Mol. Biol. 1997; 267: 418-432Crossref PubMed Scopus (53) Google Scholar). The T 2 data for N-TIMP-2 bound to N-MMP-3 are shown in Fig. 1 C. Significantly elevated T 2 values were found for Cys13 in helix 1, Lys22 in strand A, Glu57–Lys58 in the BC loop, Gly79–Gly80 in the loop between the Cys1-Cys72 disulfide bond and strand D, Gly92 in the DE loop, Ser117 in helix 2, and Gly123–Glu127 at the C terminus. In addition, the T 2 values for Gly32–Asn33 in the AB β-hairpin were also marginally above the threshold (mean plus 1 standard deviation) considered to be significant (Fig. 1 C). As observed for free N-TIMP-2, these regions of rapid internal motion were similarly identified in the 1H-15N NOE experiment (Fig. 1 D). Large negative 1H-15N NOEs were recorded for His7, Lys22, Gly32–Asn33, Glu57–Lys58, Gly92, and Tyr122–Glu127. The T 2and 1H-15N NOE data for N-TIMP-2 in the complex with N-MMP-3 are summarized in Fig. 2 B. Residues with significantly shorter T 2 values in N-TIMP-2 bound to N-MMP-3 (Fig. 1 C) were Ile19and Thr21 in strand A, Ile50, Lys51, and Phe53 at the C-terminal end of strand B, and His97, Ile98, and Leu100 in strand E and the EF loop. TheT 2 data suggest that these residues exist in two or more conformations with interconversion on the microsecond to millisecond time scale. The location of these residues are shown in Fig. 2 B. The T 1 data obtained for the complex showed the same general trends in mobility as identified by the more sensitiveT 2 and 1H-15N NOE data (data not shown). The clearest trend was a decrease inT 1 toward the C terminus of the protein (Tyr122–Glu127) indicative of rapid internal motion on a nanosecond time scale. Depressed values ofT 1 were also seen near the N terminus (His7 and Gln10), and for two glycine residues in the loop between the Cys1–Cys72 disulfide bond and strand D (Gly79 and Gly80). The average T 1 value for N-TIMP-2 was found to increase from 0.81 s for the free protein to 1.48 s in the complex. This increase is consistent with the change in molecular size of the system on complex formation resulting in a longer overall correlation time (τ m). The variation inT 1 values across the sequence for N-TIMP-2 in the complex was found to be substantially greater than that observed for the free molecule (standard deviations of 0.288 and 0.088, respectively). This greater range of T 1 values is thought to reflect the increased anisotropy of the N-TIMP-2 molecule when bound to the proteinase. The most dramatic mobility difference seen for N-TIMP-2 on N-MMP-3 binding is the loss of rapid local motion in the AB β-hairpin (Fig. 2, compare A and B). Residues Asp34, Tyr36–Asn38, and Lys41 no longer show elevated T 2 values and large negative1H-15N NOEs. In free N-TIMP-2 the regions of the protein backbone that showed greatest internal motion on a rapid picosecond to nanosecond time scale were the AB β-hairpin (Ser31–Lys41), the tight turn between strands D and E (Gly92–Asp93), and the C terminus of the protein, which shows increasing mobility from residue Gly125 onwards (Fig. 2 A). In addition, several other regions showed a more moderate degree of rapid internal motion including the N-terminal region of the protein (Ser4 and His7), the loop between strands B and C (Lys58), and the loop between the Cys1-Cys72 disulfide bond and strand D (Asp79–Lys82). The core β-barrel and the two helices of N-TIMP-2 were all found to be comparatively rigid on a picosecond to nanosecond time scale. This picture of N-TIMP-2 is consistent with that seen for other proteins, where in general the highest flexibility of the protein backbone is found in surface loop regions (29Jardetzky O. Lefevre J.-F. FEBS Lett. 1994; 338: 246-250Crossref PubMed Scopus (26) Google Scholar). The rapid motion found for the AB β-hairpin confirms our earlier suggestion that this region is flexible and able to move through a relatively large conformational space (17Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J.P. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745-11759Crossref PubMed Scopus (95) Google Scholar). This region is a very prominent feature on the surface of the protein, where it extends away form the core β-barrel into the surrounding solvent (12Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 13Tuuttila A. Morgunova E. Bergmann U. Lindqvist Y. Maskos K. Fernandez-Catalan C. Bode W. Tryggvason K. Scheider G. J. Mol. Biol. 1998; 284: 1133-1140Crossref PubMed Scopus (84) Google Scholar). A clear indication of the extent of the rapid mobility of this and other surface regions of free N-TIMP-2 is given in Fig. 3 A, where the width of the ribbon representing the backbone topology of N-TIMP-2 is scaled according to the size of the 1H-15N NOE and therefore illustrates the degree of rapid motion. Regions of high mobility in proteins are usually those least well defined in NMR structures. The average r.m.s.d values for the backbone atoms (N, C, C′) of the solution structures determined for N-TIMP-2 are shown in Fig. 4. A comparison of this graph with that for 1H-15N NOE (Fig. 1 B) reveals that the most poorly defined regions of N-TIMP-2 (i.e. highest r.m.s.d values) also show the largest negative1H-15N NOEs. In addition, there is a distinct correlation between the number of 1H-1H NOE-derived distance constraints per residue and the extent of rapid mobility. Regions of N-TIMP-2 with large negative1H-15N NOE values also showed significantly fewer than average medium and long range 1H-1H NOEs (12Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Two regions of N-TIMP-2 appear to exist in several local conformations that interconvert on a microsecond to millisecond time scale resulting in a significant reduction in their backbone amideT 2 values. The regions that show this behavior were the β-bulge at the C-terminal end of strand B (Gln49–Lys51) and the second half of strand E and the EF loop (His97–Cys101). The conformational exchange observed for Gln49–Lys51 suggests that the core β-barrel is not rigid but able to flex slightly at this point where strand B coils tightly to form the β-barrel structure (17Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J.P. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745
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