doi:10.1006/jmbi.2000.3976 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 301, 537±552

Tissue Inhibitor of Metalloproteinases-1 UndergoesMicrosecond to Millisecond Motions at Sites of MatrixMetalloproteinase-induced Fit

Guanghua Gao, Valentyna Semenchenko, S. Arumugamand Steven R. Van Doren*

Department of Biochemistry,University of Missouri, 117Schweitzer Hall, ColumbiaMO 65211, USA

E-mail address of the correspondvandorens@missouri.edu

Abbreviations used: MMP, matrixTIMP, tissue inhibitor of metallopronuclear Overhauser enhancement; Hsingle quantum coherence; CSA, chanisotropy; CPMG, Carr-Purcell-Me

0022-2836/00/020537±16 $35.00/0

The N-terminal, matrix metalloproteinase (MMP)-inhibitory fragment ofrecombinant, human tissue inhibitor of metalloproteinases (TIMP-1) exhi-bits varied backbone dynamics and rigidity. Most striking is the presenceof chemical exchange in the MMP-binding ridge reported to undergoconformational change upon MMP binding. Conformational exchange¯uctuations in microseconds to milliseconds map to the sites of MMP-induced ®t at residues Val29 through Leu34 of the AB loop and to theAla65 and Cys70 ``hinges'' of the CD loop of TIMP-1. Slow chemicalexchange is also present at the type I turn of the EF loop at the base ofthe MMP-binding ridge. These functional slow motions and other fastinternal motions are evident from backbone 15N spin relaxation at 500and 750 MHz, whether interpreted by the model-free formalism withaxial diffusion anisotropy or by the reduced spectral density approach.The conformational exchange is con®rmed by its deviation from thetrend between R2 and the cross-correlation rate Z. The magnetic ®eld-dependence indicates that the chemical exchange broadening in the ABand CD loops is fast on the time-scale of chemical shift differences. Theconformational exchange rates for most of these exchanging residues,which can closely approach MMP, appear to be a few thousand to sev-eral thousand per second. The slow dynamics of the TIMP-1 AB loopcontrast the picosecond to nanosecond dynamics reported in the longerTIMP-2 AB loop.

# 2000 Academic Press

Keywords: angiogenesis inhibitor; protein dynamics; protein-proteininteractions; magnetic ®eld-dependent relaxation; cross-correlation

*Corresponding author

Introduction

By inhibiting the activated forms of matrixmetalloproteinases (MMPs), tissue inhibitors ofmetalloproteinases (TIMPs) regulate the MMP-mediated remodeling of the extracellular matrix.This well-regulated tissue remodeling proceedsduring events of reproduction, development, andwound healing. Unbalanced regulation of MMPactivity prevails in cancer, arthritis, and other``angiogenic'' diseases (Birkedal-Hansen, 1995;

ing author:

metalloproteinase;teinase; NOE,SQC, heteronuclear

emical shiftiboom-Gill.

Nagase, 1996; Nagase & Woessner, 1999). Elevatedexpression of MMPs has been found to fostergrowth of primary and metastatic tumors(Chambers & Matrisian, 1998), in part by promot-ing angiogenesis. TIMP-1 and TIMP-2 inhibitangiogenesis in vivo (Albini et al., 1994; Johnsonet al., 1994; Moses et al., 1990; Takigawa et al.,1990). The presence of TIMPs is inversely corre-lated with oncogenicity in cultured cells (Khokhaet al., 1989). TIMP-1 suppresses tumor cell invasionin vitro (Khokha et al., 1992; Mignatti et al., 1986) asdoes TIMP-2 in culture (Albini et al., 1991). Signi®-cantly fewer and smaller metastatic tumors form inrodents once TIMPs are injected into the peritonealcavity (DeClerck et al., 1992; Khokha, 1994) or areover-expressed from a TIMP-1 transgene in strome-lysin 1 (MMP-3)-expressing transgenic mice(Sternlicht et al., 1999).

# 2000 Academic Press

538 Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1

The N-terminal 126 residue fragment of 184residue TIMP-1, known as N-TIMP-1, foldsindependently (Williamson et al., 1994a) andinhibits MMP-1, -2 and -3 with KI values of 1 to2 nM (Huang et al., 1996; Murphy et al., 1991). Thesolution structure of human N-TIMP-1 is availableat high resolution and accuracy (Wu et al., 2000).Earlier NMR structural characterization of humanN-TIMP-2, sharing 46 % sequence identity withhuman N-TIMP-1, demonstrated that the domainis organized around a ®ve-stranded b-barrel of theOB fold (Williamson et al., 1994b). Comparison oftwo X-ray structures of MMP/TIMP complexesrevealed orientation of the TIMP components withremarkably different tilts (Fernandez-Catalan et al.,1998). Superposition of the MMP catalytic domainsin the two structures, i.e. MMP-3 bound to TIMP-1(Gomis-RuÈ th et al., 1997) with MT1-MMP bound toTIMP-2, showed the TIMPs to be twisted byrotations of about 20 �, 10 �, and 5 � about orthog-onal axes intersecting at TIMP residue 4 in theMMP catalytic cleft (Fernandez-Catalan et al.,1998). Why such a large rotation of TIMPs abouttheir interfaces with MMPs occurs is an intriguingquestion. Whether the different rotations of TIMP-1and -2 about the MMP active site are in¯uenced by¯exibility in the MMP-binding ridge of TIMPs, bythe insertion of seven residues in the TIMP-2 ABloop, by plasticity in MMP active sites, and/or byeffects of crystal packing are questions that awaitfurther testing.

The AB loop of TIMPs plays a visible role inassociation with MMPs. Superposition of TIMP-2from the TIMP-2-MT1-MMP structure with theunbound TIMP-2 X-ray structure showed that ABloop residues 27-41 tilt as much as 6 or 8 AÊ uponassociation with MT1-MMP so as to make contactswith the S-shaped loop of MT1-MMP (Tuuttilaet al., 1998). This ®t of the long AB loop projectionof TIMP-2 must be facilitated by its fast internalmotions (Williamson et al., 1999). The shorter ABloop of TIMP-1 appears to draw back as much as8 AÊ from MMP-3 upon association, when compar-ing the X-ray structure TIMP-1 bound to MMP-3(Gomis-RuÈ th et al., 1997) with the NMR structureof free N-TIMP-1 (Wu et al., 2000). The comparisonshowed that the adjacent CD loop also bends backfrom MMP-3 and suggested an adjustment of theextended TIMP-1 N terminus in the middle of theMMP-binding ridge (Wu et al., 2000).

Internal motions of a protein backbone can beassessed by NMR measurements of the relaxationof amide 15N magnetization, namely transverse orR2 relaxation, longitudinal or R1 relaxation, and15N{1H} NOE. Together, these relaxation par-ameters report on equilibrium reorientation ofamide bonds, both due to overall rotational diffu-sion of the protein and due to internal motions fas-ter than the overall tumbling. The relaxation datareveal internal ¯uctuations occurring on time-scales of not only picoseconds to nanoseconds, butalso on the scale of microseconds to millisecondsvia R2. Amplitudes of the ¯uctuations of the N-H

bond vector in picoseconds to nanoseconds aresuggested by the generalized order parameterobtained by ``model-free'' interpretation of the R1,R2, and 15N{1H} NOE relaxation data (Kay et al.,1989; Lipari & Szabo, 1982a,b). Backbone dynamic¯uctuations on the picosecond to namosecond andmicrosecond to millisecond time-scales have beenidenti®ed in diverse molecular recognition surfacesof proteins (Bracken et al., 1999; Farrow et al., 1994;Hare et al., 1999; Hodsdon & Cistola, 1997;McIntosh et al., 2000; Mittermaier et al., 1999;Nicholson et al., 1995; Stivers et al., 1996; Yu et al.,1996; Zidek et al., 1999). Recently reported R2 and15N{1H} NOE data for N-TIMP-2, again 46 % identi-cal with N-TIMP-1, demonstrated that theextended AB loop of N-TIMP-2 is rich in picose-cond to nanosecond internal amide motion, whichlargely disappears once the catalytic domain ofMMP-3 is bound (Williamson et al., 1999). Protein-protein interaction surfaces known to be enrichedin microsecond to millisecond backbone motionsinclude the CD58 binding surface of CD2 (Wysset al., 1997), the CCR3 receptor binding surface of aCC chemokine (Crump et al., 1999; Ye et al., 1999),and the binding surface for kinases and phospha-tases on the bacterial response regulator Spo0F(Feher & Cavanagh, 1999). Here we present the®rst ``model-free''and reduced spectral densityanalysis of backbone relaxation of a TIMP. Mul-tiple lines of evidence, including effects of variedmagnetic ®eld and temperature, ®rmly establishthe predominance of conformational exchange, intens to hundreds of microseconds, through muchof the MMP-binding ridge of human N-TIMP-1,particularly at its sites recently shown to undergoMMP-induced ®t (Wu et al., 2000).

Results

Relaxation data and rotational diffusion

The uniformly 2H and 15N-enriched N-TIMP-1used for the 15N relaxation studies has been verystable in maintaining its 0.7 mM concentrationwithout precipitation for eight months at 20 �C andlower. Deuteration of a 14 kDa protein to 80 % wasreported to decrease amide proton line-widths by afactor of almost 2 (Markus et al., 1994). Perdeutera-tion of the aliphatic and aromatic groups of N-TIMP-1 likewise provides clearly sharper amideproton line-widths and enhanced sensitivity of itsamide-detected spectra. This sensitivity increasehas aided study of the backbone dynamics of N-TIMP-1 under conditions which increase linebroadening, i.e. low temperature which slows tum-bling and higher magnetic ®eld which exaggerateschemical exchange broadening (see below). Of 119possible 1H-15N backbone peaks, 112 were assignedin the 1H-15N HSQC spectrum (Wu et al., 1999).Due to resonance overlap or weak cross-peakintensities (see HSQC spectrum in SupplementaryMaterial), ten residues were excluded from therelaxation analysis. Thus, complete quantitative

Figure 1. Quality of ®tted (a) longitudinal and (b)transverse relaxation data for residues of N-TIMP-1 withextreme R2 relaxation rates. The relaxation data wereobtained at 20 �C at 500 MHz. Val29 and Phe73 havehigher than average R2 whereas Ala56 and Glu126 havelower than average R2 (Figure 2(b)). Symbols used are®lled squares for Val29, ®lled circles for Ala56, opensquares for Phe73, and open circles for Glu126. Mixingtimes at which duplicate measurements were made arelisted in Materials and Methods.

Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1 539

relaxation measurements were made for 102 resi-dues of free N-TIMP-1. The quality of the ®ts of R1

and R2 relaxation rates is excellent with regressioncoef®cients greater than 0.99 for most residues,over the full range of relaxation behavior (Figure 1).The residues with lowest 15N{1H} NOE, R2, andR2/R1, include Gly25, Gly53 to Ile58, Ser76,Glu125, and Glu126. Excluding these more disor-dered residues enriched in picosecond to nanose-cond motions, the average 15N{1H} NOE, expressedas Isat/Inosat, is 0.725(�0.068) at 500 MHz and0.789(�0.067) at 750 MHz (Figure 2(a)). The theor-etical maxima of 15N{1H} NOE are 0.826 at 500MHz and 0.86 at 750 MHz. Excluding the moredisordered residues, R2 values average15.1(�1.5) sÿ1 at 500 MHz and 20.7(�2.9) sÿ1 at750 MHz (Figure 2(b)) while R1 values average1.86(�0.20) sÿ1 at 500 MHz and 1.09(�0.09) sÿ1 at750 MHz. The trends of overall increases in15N{1H} NOE and R2 and decreases in R1 withspectrometer frequency agree with theoreticalexpectations.

Initial estimates of the rotational correlation timetm from R2/R1 (Fushman et al., 1994; Kay et al.,1989) averaged over the sequence were 10.3 ns at20 �C and 500 MHz, 10.8 ns at 20 �C and 750 MHz,and 13.8 ns at 8 �C and 500 MHz (Figure 2(c)). The5 % higher initial estimates of tm at 750 MHz mayhave arisen from slightly lower actual temperature.The rotational correlation time of 14.4 kDa N-TIMP-1 being higher-than-average for proteins ofsimilar size, measured at higher temperatures,prompted another hydrodynamic investigation ofpossible self-association. On a calibrated Superdex75 gel ®ltration column, N-TIMP-1 runs withapparent molecular mass of about 15.9 kDa, inde-pendent of loading concentration, suggesting itsStokes' radius to be slightly greater than that of an``ideal'' spherical monomer. Sedimentation equili-brium data were collected at N-TIMP-1 concen-trations of 50 mM, 200 mM, and 800 mM. Thesedimentation curves are best ®tted by two inde-pendent components, i.e. 95 % monomer of14.4 kDa plus species of 40 to 60 kDa present at5 %, at all three protein concentrations loaded.Since the 5 % fraction of the higher molecular massspecies is independent of N-TIMP-1 concentration,it appears not to be in equilibrium with monomerbut rather appears to be an irreversible, aggregatedheterogeneity. A well-characterized monomericprotein sharing the OB-fold with TIMP-1, namely149 residue staphylococcal nuclease, has tm of9.1 ns at 35 �C (Kay et al., 1989). The tm of staphy-lococcal nuclease is predicted to be 13.5 ns at 20 �C,given that tm/Z/T for spherical molecules, whereZ is the water viscosity of 1.00 cP (1 P = 10ÿ1 Pa s)at 20 �C and 0.71 cP at 35 �C. The tm of a proteinhaving similar size and similar degree of prolateellipsoid anisotropy to N-TIMP-1, i.e. 16 kDa HIVRNase H studied at the similar temperature of26 �C (Powers et al., 1992), can be estimated tohave tm of 12.3 ns at 20 �C. By correcting for tem-perature and viscosity differences, the tm of

14.4 kDa N-TIMP-1 is consistent with tm values ofother well-studied monomeric proteins. Whendiluted more than twofold to 0.29 mM, the 15N R2

values of N-TIMP-1 average 15.0(�1.6) sÿ1 at500 MHz and 20 �C, excluding the more disorderedresidues. The average R2 value of 15.0 sÿ1 at0.29 mM is the same as the average of 15.1 sÿ1 at0.7 mM (see plots of R2 at the two concentrationsin Supplementary Material). The rotational corre-lation time tm of 10.2 ns at 0.29 mM, estimatedfrom R2/R1, is negligibly smaller than the estimateof 10.3 ns at 0.7 mM. The lack of concentrationdependence in the R2 values further suggestsN-TIMP-1 to be monomeric. Furthermore, trendsof line broadening in the AB, CD, and EF loops(Figure 2(b) and Supplementary Material Figure 3)are unaffected by dilution. It appears quite unlikelythat there is any reversible monomer to oligomerequilibrium to contribute local line-broadeningeffects in N-TIMP-1.

Figure 2. 15N NMR relaxation parameters for human N-TIMP-1 at 20 �C at 500 and 750 MHz spectrometer frequen-cies. Red circles indicate results at 750 MHz. Black squares indicate results at 500 MHz. (a) 15N{1H} NOE, (b) R2 withtcp � 892 ms, and (c) R2/R1 are plotted as a function of residue number. Error bars for R2 of many residues are withinthe symbol size. Locations of a-helices are marked with cylinders and locations of b-strands with arrows.

540 Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1

Confirmation of exchange broadeningevidence of micro- to millisecond motion

Carefully interpreted line-broadening effectsof chemical exchange processes, i.e. the Rex

contribution to R2, reveal micro- to millisecond¯uctuations in amide environments of potentialfunctional relevance. R2/R1 ratios can, however,increase not only by chemical exchange (Rex) con-tributions to R2, but also by orientation of N-Hbond vectors nearly parallel with the long axis ofthe protein's anisotropic diffusion tensor (Tjandraet al., 1995). Use of high R2/R1 ratios to identifysites of micro- to millisecond conformational

exchange then demands some caution regardingeffects of amide bond orientations given that therotational diffusion of N-TIMP-1 is anisotropic (seebelow). Plotting R2/R1 against a measure of amidebond orientation, namely the residual dipolarcoupling between amide proton and nitrogen in aweakly oriented sample, enables discrimination ofincreases in R2/R1 due to conformational exchangefrom increases due to amide bond orientation(de Alba et al., 1999). Such a plot of the R2/R1

values of N-TIMP-1 plotted against previouslymeasured dipolar couplings (Wu et al., 2000) isshown in Figure 3(a). The upward deviation fromthe correlation con®rms those residues as having

Figure 3. Con®rmation of micro-to millisecond exchange broadeningin R2 values of human N-TIMP-1.(a) R2/R1 at 750 MHz and 20 �C isplotted against residual dipolarcoupling 1DNH for each backboneamide having 15N{1H} NOE (Isat/Inosat) greater than 0.65 as pre-scribed (de Alba et al., 1999). Theresidual dipolar coupling measure-ments of a weakly Pf1 phage-oriented sample were describedrecently (Wu et al., 2000). Residueswith elevated R2/R1 independentof amide bond orientation arelabeled and colored red. (b) Amidenitrogen cross-correlation rate Zplotted against R2 using tcp � 892ms at 500 MHz and 8 �C. Residuesexperiencing high-amplitude, pico-to nanosecond internal amide re-orientation are numbered andmarked with blue triangles. Resi-dues with R2/Z greater thanexpected from the linear correlationbetween Z and R2, stronglysuggesting micro- to millisecondexchange broadening, are num-bered and marked with red circlesand error bars.

Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1 541

R2/R1 heightened by chemical exchange. Forexample, the R2/R1 of Thr32 is no greater than thatof residues with DNH values of ÿ13 to ÿ15 Hz thatlie near the regression line (Figure 3(a)). However,Thr32 lies above the trend of lower R2/R1 expectedfor its DNH value of 8 Hz, suggesting that its linebroadening arises from genuine chemicalexchange.

The presence of microsecond to millisecond con-formational exchange of AB loop residues, such asThr32, is further corroborated by relaxation datacollected at the low temperature of 8 �C. Thedecrease in temperature is expected to increase Rex

at sites having kex > 3.2/tcp, where tcp is the spa-cing between p pulses of the CPMG train of thepulse sequence of the R2 measurement (Mandelet al., 1996). With Rex thus enhanced by low tem-perature, cross-correlation rates were measured(Tjandra et al., 1996) and plotted against the R2

values measured at 8 �C (Figure 3(b)). The S2

measure of backbone rigidity increases (see below)progressively up the trend line from lower left toupper right in Figure 3(b). The cross-correlationrate Z and R2 depend similarly on J(0) and J(oN)but differ in that Z lacks Rex contributions. Conse-quently, amide nitrogen atoms with signi®cant Rex

contributions to R2 deviate from the linear corre-lation between Z and R2 (Fushman & Cowburn,1998). Thus, the combination of low temperatureand Z versus R2 (Figure 3(b)) tease out the subtleline broadening due to conformational exchangenow evident for all AB loop residues Val29through Leu34, in addition to the sites of exchangein the CD and EF loops already evident.

Since the Rex line broadening from exchangewhich is fast on the chemical shift scale is pro-portional to the square of the chemical shift differ-ence between states (Luz & Meiboom, 1963), Rex isexpected to be proportional to the square of themagnetic ®eld Bo of the spectrometer. The spectral

542 Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1

density at zero frequency, Jeff(0), is sensitive tosuch chemical exchange and increases with Bo atsites undergoing exchange (Peng & Wagner, 1995).Because Jeff(0) also decreases at sites of increasingmobility on the pico- to nanosecond scale, it pro-vides an appealing summary of backbone amidereorientational dynamics probed by 15N relaxation(Peng & Wagner, 1995; Wyss et al., 1997).Figure 4(a) shows Jeff(0) derived from R2, R1 and15N{1H} NOE collected at 500 and 750 MHz.Elevation of Jeff(0) above the median values ofaround 4 to 5 ns/radian throughout the regions ofsecondary structure strongly suggests the presenceof slow micro- to millisecond chemical or confor-mational exchange. Consistent with elevated R2/R1

(Figures 2(c)and 3(a)) and high R2/Z (Figure 3(b)),high values of Jeff(0) implicate AB loop residuesVal29 through Leu34; CD loop residues Ala65,Cys70, Phe73 through Arg75; and EF loop residuesIle96 and Thr97 as the most obvious sites of micro-to millisecond ¯uctuations (Figure 4(a)). The750 MHz results additionally suggest Tyr38 andGlu82 (Figures 2(c), 3(a) and 4(a)) to be sites ofmicro- to millisecond exchange not evident at500 MHz. The marked increases in Jeff(0) at 17.6 Trelative to 11.7 T at (Figure 4(a)) for residues Val29through Leu34, Tyr38, Ala65, Cys70, Phe73through Arg75, and Glu82 suggest their exchangeis fast on the chemical shift scale. Subtle increasesin Jeff(0) with magnetic ®eld Bo can be seen else-where such as at sites in the C-terminal helix andjust beyond (Figure 4(a)). In contrast, lack ofincrease of Jeff(0) with Bo implies that Ile96 andThr97 fail to obey two-site fast exchange behavior.

Model-free analysis

Prior to model-free interpretation of the 15Nrelaxation of each residue (Clore et al., 1990; Kayet al., 1989; Lipari & Szabo, 1982a,b), anisotropy inthe rotational diffusion of N-TIMP-1 was esti-mated. The ratio of the principal moments of iner-tia of N-TIMP-1 is 1.00:0.97:0.62, indicating that N-TIMP-1 is a prolate ellipsoid. Using the R2/R1

ratios of 70 selected residues and coordinates ofthe NMR structure of N-TIMP-1 (PDB accessioncode 1d2b, model 22), components of an axial dif-fusion tensor, Dk/D?, were estimated. A model ofdiffusion with axial symmetry confers a statisti-cally signi®cant reduction in w2, relative to use of asingle rotational diffusion constant. The startingvalues of the parameters tm and Dk/D? in theModelFree analysis of the relaxation data were10.3 ns and 1.19 at 20 �C, 500 MHz; 13.8 ns and1.13 at 8 �C, 500 MHz; 10.8 ns and 1.13 at 20 �C,750 MHz.

For model-free ®ts, the 500 MHz, 20 �C relax-ation data were supplemented with 750 MHz R1

values while the 750 MHz data were supplementedwith 500 MHz R2 values. Inclusion of a fourthrelaxation parameter from another ®eld enabled Ftesting of the suitability of dynamical models 4and 5, which involve ®tting of three parameters

(Table 1). At 500 MHz and 8 �C, the standard R1,R2 and NOE relaxation data were ®tted (see Sup-plementary Material). Models were selected by F-test (Mandel et al., 1995), except for the AB loop at20 �C and 500 MHz. Since the AB loop clearlyexperiences conformational exchange throughout(Figures 2(b) and (c), 3(a) and (b)), models 3 or 4which employ the Rex term were used throughoutthe AB loop for all three data sets. This permittedestimation of Rex as a function of ®eld and tem-perature. The model selection strategy yielded thesame choice of motional models for all three datasets for 68 of the 102 residues, i.e. for the data col-lected at 20 �C and 8 �C at 500 MHz as well as at20 �C at 750 MHz. The differences in model selec-tion for the other 34 residues, among the three con-ditions of ®eld and temperature, involved theaddition or removal of just one parameter. Com-paring the ®ts of 500 MHz data at 20 �C and 8 �C,only 14 residues were assigned different models.Comparing the ®ts of data collected at 20 �C at500 MHz and 750 MHz, 74 residues were ®ttedwith the same choice of models. Rex terms (model3 or 4 in Table 1) are required for good ®ts of therelaxation of 15 residues at 500 MHz and 36 resi-dues at 750 MHz. Summaries of choice of Model-Free spectral density models and resultingparameter ®ts are reported in SupplementaryMaterial. Final values for tm and Dk/D? obtainedafter three rounds of ModelFree optimization were10.32(�0.05) ns and 1.13(�0.04) at 20 �C, 500 MHz(�R1 at 750 MHz); 14.1(�0.08) ns and 1.15(�0.05)at 8 �C, 500 MHz; 10.87(�0.05) ns and 1.21(�0.04)at 20 �C, 750 MHz (�R2 at 500 MHz).

The values of S2, the square of the generalizedorder parameter, determined from the relaxationdata at 500 MHz (�R1 at 750 MHz) and at750 MHz (�R2 at 500 MHz) agree within theuncertainty for most of the residues (Figure 4(b)and (c)). The average values of S2 are above 0.95for most residues in the a-helices and b-strands forall three data sets. Much smaller S2 values prevailat b-strand A residues Gly25 and Thr26, BC loopresidues Leu52 through Ile58, Ser76 of the CDloop, and C-terminal Glu125 and Glu126(Figure 4(b) and (c)), all of which require model 5(Table 1) for good ®ts. The values of S2 for thesecomparatively disordered residues are systemati-cally higher at 750 MHz if only three parameters ata single ®eld are ®tted. Addition of a fourth par-ameter measured at a second ®eld brings the S2

values of these residues, derived from sup-plemented 500 MHz and 750 MHz data, to goodagreement. Modest dips in S2 occur at the AB loop;at CD loop residues Ala65, Cys70, Arg75, andAsn78; and at Leu89, Asp91, and Val102. Thesubtle drop in S2 of these residues is more appar-ent from model-free ®ts of 500 MHz data thanfrom ®ts of 750 MHz data (Figure 4(b) and (c)).The relative dips in 15N{1H} NOE measured at750 MHz for these residues being less than at 500MHz (Figure 2(a)) may account for this smallsystematic discrepancy.

Figure 4. Reduced spectral density and ModelFree analysis of 15N relaxation at 500 and 750 MHz. Red circles indi-cate results at 750 MHz. Black squares indicate results at 500 MHz. (a) Jeff(0) (Farrow et al., 1995; Peng & Wagner,1995). Several residues subject to micro- to millisecond exchange are labeled. (b) Generalized order parameter S2

derived from 500 MHz relaxation data. (c) S2 derived from 750 MHz relaxation data. (d) Rex exchange broadeningderived from model-free ®ts of 500 and 750 MHz data.

Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1 543

15N chemical exchange contributions (Rex) to theapparent transverse relaxation rate constants wereobserved for 15 residues at 20 �C, 500 MHz(Figure 4(d)) and for 16 residues at 8 �C, 500 MHz(not shown), clustered at the AB loop (Val29-Gln36), the CD loop (Ala65, Cys70, and Phe73-Arg75), and the EF loop (Ile96,Thr97, and at 8 �CThr98). Among the 21 additional residues requiringmodest Rex terms for improved model-free ®ts of750 MHz relaxation data, conserved residues T63,G71, C99, plus S100 lie at or near the base of the

MMP-binding ridge, adjacent to residues with lar-ger Rex. Conserved residues 7, 9, 10, 112, 115, 120,and less-conserved 119 which contact the C-term-inal lobe in full-length TIMP-1 (Gomis-RuÈ th et al.,1997) also require Rex terms for satisfactory ®ts of750 MHz data, but not 500 MHz data (Figure 4(d)).Glu82 and conserved Arg20 which lie at the con-served positive patch (Wu et al., 2000) require Rex

just in the 750 MHz model-free ®t. In the case oftwo-site fast chemical exchange, Rex,750MHz/Rex,500MHz is predicted to equal (750/500)2 or 2.25.

Table 1. Models used to ®t experimental relaxation data to the extended Lipari-Szaboform of the spectral density function

ModelFit

parameters Spectral density function

1 S2 J(o)�2/5{(S2tm)/[1�(otm)2]}2 S2, te J(o)�2/5{(S2tm)/[1�(otm)2]�[(1-S2)t]/[1�(ot)2]}3 S2, Rex J(o)�2/5{(S2tm)/[1�(otm)2]}4 S2, te, Rex J(o)�2/5{(S2tm)/[1�(otm)2]�[(1-S2)t]/[1�(ot)2]}5 Ss

2, Sf2, te J(o)�(2Sf

2/5){(Ss2tm)/[1�(otm)2]�[(1ÿSs

2)t]/[1�(ot)2]}

See references for details of the model-free formalism (Lipari & Szabo, 1982a) and its extension(Clore et al., 1990). In the equations above, t � tetm/(te � tm), tm is the isotropic rotational correla-tion time of the molecule and te is the effective correlation time for internal motions. S2 � Sf

2Ss2 is

the square of the generalized order parameters, characterizing the amplitude of the internalmotions. Sf

2 and Ss2 are the squares of the order parameters for the internal motions on the fast and

slow time-scales, respectively.

544 Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1

Rex,750MHz/Rex,500MHz lies within one standard devi-ation of 2.25 for residues Val29, Gln31 throughTyr35 and Phe73 through Arg75 (Figure 4(d)). ForAla65 and Cys70 which have broader line-widthsand Rex of greater uncertainty, Rex,750MHz/Rex,500MHz

lies within 1.5 standard deviations of 2.25. In con-trast, EF loop residues Ile96 and Thr97 fail to obeythe fast exchange relationship (Figure 4(d)).

Estimation of time-scale ofconformational exchange

Approximate ranges for the chemical exchangerate kex can be estimated on the basis of tempera-ture dependence of their Rex values, as demon-strated in a pioneering study of temperature-dependent dynamics of ribonuclease H (Mandelet al., 1996). The authors showed that kex < 3.2/tcp

when increasing temperature increases Rex andthat kex > 3.2/tcp when increasing temperaturedecreases Rex, where tcp is the spacing between ppulses of the CPMG train of the R2 pulse sequence.To further narrow the ranges of kex values of N-TIMP-1, in-phase R2 values were measured at 8 �Cand 20 �C using different lengths of tcp. The tem-perature range was restricted by the need to staybelow room temperature to maintain solubility of

Table 2. Estimates of ranges of chemical exchange rates

Residueln(Rex, 20 �C/Rex, 8 �C)

tcp � 642 msln(Rex, 20 �C/R

tcp � 89

Val29 0.89 ÿ0.10Gln31 0.59 0.09Thr32 ÿ0.30 ÿ0.19Thr33 0.97 0.47Leu34 ÿ0.25 ÿ0.05Ala65 2.22 0.70Phe73 0.43 ÿ0.08His74 ÿ0.13 ÿ0.50Arg75 0.41 ÿ0.02Ile96 0.95 0.29

Estimates of kex were obtained from dependence of Rex upon te500 MHz (Mandel et al., 1996).

a Rex values at tcp � 892 ms have been averaged from model-free ®

N-TIMP-1. Predicted Boltzmann populationchanges from 8 �C to 20 �C are too small to affectestimates of kex noticeably. The R2 values acquiredwith varied tcp delays at both 8 �C and 20 �C wereused in model-free analyses to obtain Rex valuesfor each combination of tcp and temperature. Sinceln(Rex,20 �C/Rex,8 �C) < 0 implies kex > 3.2/tcp andln(Rex,20 �C/Rex,8 �C) > 0 implies that kex < 3.2/tcp, theresults for tcp values of 642 ms, 892 ms, and 1142 ms(Table 2) permit estimation of kex relative tobounds of �5000, 3600, and 2800 sÿ1, respectively.Minimal temperature dependence of Rex such thatÿ0.15 < ln(Rex,20 �C/Rex,8 �C) < 0.15 suggests that kex

is not clearly different from 3.2/tcp. The estimatesof kex fall between 2800 sÿ1 and 5000 sÿ1 for anumber of exchanging residues (Table 2). Thr32and Leu34 appear to have kex > 8200 sÿ1, sincetheir Rex values are greater at lower temperaturefor shorter tcp of 392 ms.

Discussion

Backbone rigidity and its possible implications

Most of the backbone of human N-TIMP-1 exhi-bits a striking lack of internal motion on the picose-cond to nanosecond scale. The generalized order

ex, 8 �C)a

2 msln(Rex, 20 �C/Rex, 8 �C)

tcp � 1142 msApproximate range of kex

(sÿ1)

ÿ0.27 2800-5000ÿ0.22 2800-5000ÿ0.70 >8200ÿ0.40 2800-5000ÿ0.22 >8200

1.17 <2800ÿ0.31 2800-5000ÿ0.47 >3600ÿ1.34 2800-5000ÿ0.37 2800-3600

mperature and tcp delay of CPMG experiments performed at

ts of duplicate series of CPMG spectra.

Figure 5. Locations of clearest backbone ¯uctuations of human N-TIMP-1 occurring in micro- to milliseconds(yellow) and in pico- to namoseconds (red). Residues with S2 greater than 0.65 are colored blue. Residues whererelaxation data are not available, because their amide peaks are overlapped or missing, i.e. proline residues or unas-signed, are colored gray. Sites of distinctive dynamics are labeled. CD loop residues M66 through V69 whose peaksare broadened beyond detection are colored off-white. (a) The solution structure of N-TIMP-1 (PDB accession code1d2b, model 22) is represented with a backbone tube of thickness proportional to the backbone RMSD (Wu et al.,2000). (b) Representative NMR model 3 of free N-TIMP-1 (cyan, grey, yellow, and red ; PDB code 1d2b) is superim-posed with the X-ray model of the complex of TIMP-1 with the catalytic domain of MMP-3 (blue; PDB code 1uea)(Gomis-RuÈ th et al., 1997). MMP-3 is marked by a dot representation of its molecular surface with its catalytic zinc ionin yellow dots.

Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1 545

parameter S2 (Figure 4) averaged over most resi-dues is 0.95, excluding the residues with highestamplitude fast internal motions, namely G25, BCloop residues 53-58, S76, and the C terminus. Theregions of high and low S2 correlate well with theregions of low and high backbone RMSD in thesolution structure of N-TIMP-1 (Figure 5(a)), con-®rming the integrity of the solution structurere®nement of N-TIMP-1 (Wu et al., 2000).

Cys3 provides one of the few amide peaks avail-able for reporting on the backbone mobility of theextended N terminus of N-TIMP-1 which insertsinto the active-site cleft of the MMP (seeFigure 5(b)). The S2 value of 0.90 for Cys3 at 20 �C(Figure 4(b) and (c)) suggests the amide bond ofCys3 to be rather restricted from fast internal reor-ientation, despite its high exposure to solvent. ItsS2 value can suggest the amplitude of its internalamide bond reorientation when interpreted using areasonable model of the internal motion (Lipari &Szabo, 1982b) such as wobbling diffusion within acone of semi-angle ymax (Kinosita et al., 1977; Lipari& Szabo, 1980; Richarz et al., 1980) where:

ymax � cosÿ1�1=2��1� 8Scone�1=8 ÿ 1�� �1�Scone

2 of 0.90(�0.03) for Cys3 suggests the maxi-mum excursion of the Cys3 amide bond from theaxis of a cone, i.e. ymax to be around 15(�2.5) �. Aninternal correlation time te of Cys3 of a few hun-dred picoseconds, estimated by model-free ®tting,is consistent with the modestly elevated values ofreduced spectral densities for Cys3 at high frequen-cies of 75 to 750 MHz (Supplementary Material).The disul®de cross-linking of Cys1 and of Cys3 tothe OB-fold underneath must confer a signi®cant

degree of rigidity otherwise unexpected of such ahighly exposed N-terminal ridge. The N-terminalfast dynamics being moderated by the disul®debonding could minimize the energetic cost of lossof con®gurational entropy upon its insertion intoan MMP active site. Consistent with this hypoth-esis, disruption of the Cys1 to Cys70 disul®deraises the KI of N-TIMP-1 for MMP-1, -2, and -3 bythree orders of magnitude (Huang et al., 1997).Once TIMP-1 binds an MMP, using the amino andcarbonyl groups of Cys1 to coordinate the catalyticzinc in the MMP active site (Gomis-RuÈ th et al.,1997), further rigidi®cation of the N terminus ofTIMP-1 is likely.

Another OB-fold protein in its unbound state,namely the SN form of staphylococcal nuclease,was also reported to have very high S2 throughmost of its backbone (Alexandrescu et al., 1996).Once staphylococcal nuclease forms a ternary com-plex with Ca2� and thymidine bisphosphate, aform known as SN-T, it has lower S2 through mostof its backbone (Alexandrescu et al., 1996; Kay et al.,1989), suggesting that the ligand af®nity is coupledto favorable backbone conformational entropyincrease upon association. The binding of single-stranded DNA to a topoisomerase I also causeswidespread decreases of backbone S2 (Yu et al.,1996). The concept of coupling of ligand bindingaf®nity with favorable entropy increase, potentiallywidespread through the protein backbone, hasbeen articulated (Zidek et al., 1999). Turning to aprotein-protease interaction, leech carboxypepti-dase inhibitor (LCI) has uniformly high 15N{1H}NOEs of �0.74 through most of its rigid backbone.In three loops of LCI, the NOE values drop to 0.5

546 Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1

to 0.67 upon binding to carboxypeptidase(Reverter et al., 2000), suggesting a favorable netincrease in backbone entropy upon association.The high af®nity of the catalytic domain of humanstromelysin (MMP-3) for human N-TIMP-1 is dri-ven by favorable entropy change (unpublishedresults). Whether there could be a net favorableincrease in TIMP-1 con®gurational entropy uponMMP binding, to provide part of the favorableentropy of association, remains an intriguingquestion.

Sites of micro- to millisecond scale motion

The Cys1-Cys70 disul®de of TIMP-1 and theCys14-Cys38 disul®de of BPTI are similar in thatthey both appear to rigidify loops that insert intoprotease active sites for tight inhibition. Confor-mational exchange in micro- to milliseconds pre-vails in disul®de-linked protease-binding loops ofboth BPTI (Beeser et al., 1997; Szyperski et al., 1993)and TIMP-1. Most of the sites in N-TIMP-1 display-ing micro- to milliseconds chemical exchangeeffects at 500 MHz can be readily correlated withfunctional regions of the MMP-binding ridge ofTIMP-1. These sites of slow motion are con®rmedby multiple lines of evidence including distinctiveelevation of R2/R1, Rex, and Jeff(0) by high magnetic®eld or by low temperature (Figures 2, 3 and 4).The collection of such experiments identify the fol-lowing MMP-binding regions as being enriched inmicrosecond to millisecond motions: AB loop resi-dues Val29 through Leu34; CD loop residuesAla65 and Cys70; and EF loop residues I96, T97,and perhaps also Thr98 to Ser100 (Figure 5(b)).Judging from the X-ray structure of the complex ofTIMP-1 with the catalytic domain of MMP-3 (stro-melysin) (Gomis-RuÈ th et al., 1997), several of theresidues of the AB, CD, and EF loops of TIMP-1most clearly undergoing slow ¯uctuations can clo-sely approach the MMP (Figure 5(b)). In the ABloop, Val29 of TIMP-1 comes within 6 AÊ of N-terminal Thr85 and Phe86 of MMP-3 while Leu34of TIMP-1 approaches within 4 AÊ of Phe154 ofMMP-3. In the CD loop, Ala65 of TIMP approacheswithin 4 AÊ of Phe86 of MMP-3 and Cys70 ofTIMP-1 approaches within 4 AÊ of Val163 of MMP-3. TIMP-1 residues Met66 through Val69, whichapproach within 4 AÊ of MMP-3 in the crystal, pre-sumably undergo slow motion as well (off-white inFigure 5(b)), since their peaks are broadenedbeyond detection. The type I turn residues Thr97to Ser100 of the EF loop of TIMP-1, underneath thecritical N terminus, all may undergo micro- tomilliseconds exchange, when considering the Rex

made evident by low temperature and high ®eld of17.6 T. Cys99 (Rex at 750 MHz only) and Thr97each approach within 5 AÊ of Asn162 and Val163 ofMMP-3 in the crystal structure. Thr98, having Rex

at 8 �C, comes within 6 AÊ of Val163 of MMP-3 inthe complex. Ser100, with Rex at 750 MHz, canapproach within 5 AÊ of Asn162 in the complexwith MMP-3. The slow, temperature-sensitive

mobility of the MMP-binding ridge of TIMP-1accommodates the hypothesis that the disparity inTIMP orientations in crystals of TIMP-MMP com-plexes (Fernandez-Catalan et al., 1998) could bedue to the ¯exibility inherent in the MMP-bindingridge of TIMPs (see Introduction).

The variable TIMP orientation with respect tothe MMP active site may also be related to differ-ing ¯exibility of MMP active sites. TIMP-1 residuesThr2 to Val4 occupy positions at or near MMP sub-sites S10 to S30 (Gomis-RuÈ th et al., 1997), which pro-gress to the right from the catalytic zinc inFigure 5(b). Parts of subsites S10 to S30 of MMP-1(collagenase 1) and MMP-3 (stromelysin 1) exhibita plasticity evident both in backbone dynamics(Moy et al., 1997; Yuan et al., 1999) and in structur-al variability in these subsites among numerousstructures of MMP-inhibitor complexes. At the per-iphery of the S30 subsite of MMP-3, residues 191and 192, and perhaps 223 and 224, become morerigid once an inhibitor is bound at the S10 to S30subsites (Yuan et al., 1999). In contrast, in the loopbelow the S10 to S30 subsites, MMP-1 residues 138-144 (corresponding to MMP-3 residues 220-226)manifest disorder with S2 < 0.6 for MMP-1 bothfree and with inhibitor bound near the S10 to S30region (Moy et al., 1997). Since MMPs can vary inthe dynamic character of their S10 to S30 pockets,insertion of the N terminus of TIMPs in this region(at the right in Figure 5(b)) might subtly differ.Plasticity around the S10 to S30 subsites of theMMP might help accommodate docking of thedifferent residues found at positions 2 (Thr, Ser)and 4 (Val, Ala, Ser) of native TIMPs. Most of theS1 to S3 subsite region to the left of the zinc inFigure 5(b) is instead consistently rigid, with S2

values averaging 0.89 to 0.86 whether inhibitor isbound to the left or right side of the catalytic cleft(Yuan et al., 1999). In the S-shaped loop of MMP-3,however, residues 150 to 155 which roof the S1 toS3 pockets do become more rigid on inhibitorbinding at the S1 to S3 pockets at the left (inFigure 5(b)), with S2 values increasing from 0.75 to0.85 (Yuan et al., 1999).

Docking of TIMPs to the rigid left side of MMPsseems to demand that the TIMP AB and CD loops¯ex to ®t these rigid S1 to S3 pockets (Fernandez-Catalan et al., 1998; Wu et al., 2000). The MMP-inducible ®t of the AB and CD loops correlateswith these loops' thermally activated exchangeprocesses occurring in tens to hundreds of micro-seconds (see Figures 2 to 5 and Table 2). Where anexchange rate kex is suf®ciently far from 3.2/tcp,ln(Rex,20 �C/Rex,8 �C) provides an Arrhenius estimateof the apparent activation barrier (Mandel et al.,1996). The larger ln(Rex,20 �C/Rex,8 �C) values of 0.4to 1.3 seen in Table 2 correspond to crude esti-mates of activation barriers ranging from 23 to74 kJ/mol. Similar approximate activation barriersto chemical exchange of about 20 to 52 kJ/molwere reported for 18 residues of Escherichia coliRNase HI, from temperature dependence at asingle tcp delay (Mandel et al., 1996). For compari-

Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1 547

son, NMR studies of small model compoundsrevealed activation barriers for methyl rotationsand for disul®de isomerization. Such methylrotations involve low activation barriers of 10 to18 kJ/mol (Kowalewski, 1991). Disul®de cis-transisomerization encounters barriers of 29 to 38 kJ/mol (Fraser et al., 1971), of possible relevance to theCD and EF loops of TIMP-1, since Cys70 (CD) islinked to Cys1 and Cys99 (EF) is linked to Cys3.Higher barriers of 57 to 66 kJ/mol were observedfor impeded aromatic ring ¯ips in BPTI (Wagneret al., 1987; Wagner & WuÈ thrich, 1978). A still high-er barrier of about 90 kJ/mol was reported in BPTIfor the exchange of a buried water molecule(Denisov et al., 1996). The crude estimates of bar-riers to the backbone chemical exchange of humanTIMP-1, provided by Table 2, span this full rangeof diverse activation barriers.

Some residues such as Tyr38, Glu82, and Phe73through Arg75 of N-TIMP-1 show clear evidenceof chemical exchange, but are located outside theMMP-binding ridge. Phe73 through Arg75 appearto form a solvent-exposed exposed buttress of theMMP binding ridge. Several N-terminal and C-terminal residues of N-TIMP-1 manifest small Rex

values only at 750 MHz. Of these, conserved Gln9,Thr10, Gln112, Gly115, Tyr120, plus little con-served Thr119 cluster to form the internal interfacebetween the N-terminal domain of TIMP-1 and theC-terminal lobe. The subtle exchange broadeningof these residues may be an artifact of the trunca-tion of the C-terminal lobe of TIMP-1.

Comparison with N-TIMP-2 dynamics

The most striking difference in dynamicsbetween N-TIMP-2 and N-TIMP-1 is that the ABloop of the former is rich in high-frequency (pico-to namosecond) internal motion, most evidentfrom 15N{1H} NOE values but also from T2 values(Williamson et al., 1999). These fast dynamics ofthe AB loop of TIMP-2 are easily understood fromit being seven residues longer and much more sol-vent exposed than the AB loop of TIMP-1. Com-parison of free and MMP-bound structures ofTIMP-1 and -2 show that in both cases the AB loopexperiences induced-®t on MMP binding. The ener-getics and binding contacts of the AB loops ofTIMP-1 and TIMP-2 must differ. The longer ABloop of TIMP-2 presents more residues for favor-able contact with MMP than does the much shorterAB loop of TIMP-1 (Fernandez-Catalan et al., 1998).Yet the longer AB loop of TIMP-2 may alsoencounter the higher energetic cost of greater lossof con®gurational entropy on association withMMP.

Other dynamical differences between N-TIMP-1and -2 are found more remote from the MMP-bind-ing ridge. The dip in R2 and S2 for Gly25 andThr26 of the Gly25 to Pro27 interruption ofb-strand A are unique to N-TIMP-1 and mightpromote the mobility in microseconds of the shortAB loop nearby. The micro- to millisecond chemi-

cal exchange broadening reported for N-TIMP-2b-strand B residues Gln49 and Lys51 (Williamsonet al., 1999) is not observed at the equivalent resi-dues Met42 and Lys44 of N-TIMP-1. The highrigidity throughout this part of b-strand B ofN-TIMP-1 is evident in its high S2 values andabsence of Rex (Figure 4), casting some doubt onthe recent suggestion that this part of b-strand B is¯exible (Williamson et al., 1999). More plausible isthe hypothesis that the Ala21Thr substitution inthe N-TIMP-2 studied promotes the chemicalexchange at neighboring Gln49 and Lys51. Pointmutations have been shown to introduce consider-able chemical exchange broadening in the spectraof BPTI (Beeser et al., 1997) and calmodulin(EvenaÈs et al., 1999). In the BC loop, N-TIMP-2 hasone residue, Lys58, with pico- to namosecondinternal motion (Williamson et al., 1999) whereasthe ®ve residue insertion in the BC loop of N-TIMP-1 results in the six residues from Gly53through Ile58 having fast internal motion(Figures 2, 3(b) and 4). Next to the BC loop in thetight turn between b-strands D and E, Gly92 andAsp93 of N-TIMP-2 exhibit fast internal motionthat the equivalent Gln90 and Asp91 of N-TIMP-1do not.

Pertinent to their inhibition of MMPs, N-TIMP-1and -2 share moderate pico- to namosecond back-bone mobility of the N-terminal ridge, the micro-to millisecond chemical exchange in the EF loopunderneath the N terminus, and the total broaden-ing of amide peaks of CD loop residues Glu67 toVal69 (Ser68 to Val71 in TIMP-2) which contactMMP. The missing peaks of the residues of the CDloops of both proteins are presumably broadenedaway by micro- to millisecond chemical exchange.For N-TIMP-1, this assumption is further corrobo-rated in that the largest measured exchange-mediated line broadening occurs at Cys70 andAla65 which ¯ank this segment with peaks broa-dened beyond detection (Figures 2, 3 and 4).

Features of sites of micro- tomillisecond motion

A characteristic of most of the residues of N-TIMP-1 which ¯uctuate in micro- to milisecond isthat they lack backbone hydrogen bonds, judgingfrom the high-resolution solution structure ofhuman N-TIMP-1 (Wu et al., 2000). Most of theexceptions are residues hydrogen-bonded toanother exchanging residue, i.e. Thr32 to the Asn30side-chain, Leu34 to Ala65, Thr97 to Ser100, andGly115 to Tyr120. The long CD loop, with its sev-eral residues undergoing micro- to milisecondexchange, is quite hydrophilic and exposed at thesurface. The hydrophilic character of the CD loopis consistent with the hydrophilic character of theequivalent long loop joining the third and fourthb-strands in other OB-fold proteins, of no obviousrelation to one another in sequence or function(Alexandrescu et al., 1999). The beginning of thishydrophilic loop connecting the third and fourth b-

548 Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1

strands of OB-fold proteins, e.g. Ala65 to Cys70 inTIMP-1, appears to contribute to or lie near themolecular recognition surface of most of thesediverse proteins (Murzin, 1993), certainly includingTIMPs. Since pronounced line broadening alsooccurs in the beginning portion of the equivalentloop after the third b-strand of staphylococcalnuclease (Alexandrescu et al., 1996; Kay et al.,1989), micro- to millisecond dynamics of thishydrophilic recognition loop could prove to be afeature shared widely among diverse members ofthe OB-fold.

In summary, N-TIMP-1 projects from a remark-ably rigid OB-fold scaffold a ridge of varied dyna-mical character for inhibition of MMPs. The ridgefeatures: (1) an N terminus with fast backbone¯uctuations of amplitude limited by disul®decross-linking; (2) an EF loop underneath ¯uctuat-ing much more slowly on a micro- to millisecondscale; (3) the adjacent CD loop with severalresidues undergoing micro- to millisecond confor-mational exchange; and (4) the peripheral AB loop¯uctuating in tens to hundreds of microseconds.The slow motion throughout the AB loopcorrelates remarkably well with the sizeableMMP-induced ®t reported throughout the loop(Wu et al., 2000). The slow motion of CD loopresidues Ala65 and Cys70 (and presumably forMet66-Val69 whose peaks are broadened beyonddetection) coincides with the modest MMP-induced bending about Pro64/Ala65 and Cys70(Wu et al., 2000). The pronounced line broadeningin the functional CD loop may prove to be afeature common among diverse members of theOB-fold, such as the well-characterized staphylo-coccal nuclease (Alexandrescu et al., 1996; Kay et al.,1989).

Materials and Methods

Sample preparation

The N-terminal domain of recombinant human TIMP-1 was expressed in E. coli harboring the pET3a-N-TIMP1vector, generously provided by Keith Brew, and puri®edfrom inclusion bodies (Huang et al., 1996). The 15N-enriched, perdeuterated protein was expressed in a med-ium which was 50 % M9 minimal medium, containing99 % 2H2O and 15NH4Cl, mixed with 50 % Celtone-DN(Martek Biosciences, Columbia, MD). NMR spectra wererecorded on solutions (650 ml in an 8 mm Shigemi micro-cell or 350 ml in a 5 mm Shigemi microcell) containing�0.70 mM protein at pH 6.0 with 20 mM deuteratedsodium acetate, 150 mM NaCl, 1 mM NaN3 and 7 %2H2O. In order to maintain its solubility at high concen-tration, N-TIMP-1 was studied at moderate temperaturesof 20 �C and below.

Hydrodynamics

The apparent molecular mass of N-TIMP-1 was esti-mated using a Superdex 75 HiLoad 16/60 gel ®ltrationcolumn (Amersham Pharmacia Biotech, Inc.). The col-umn was calibrated using bovine serum albumin(67 kDa), ovalbumin (43 kDa), chymotrypsin (25 kDa),

cytochrome c (12.3 kDa), and aprotinin (6.5 kDa).A single exponential decay ®t the ®ve standards withthe excellent regression coef®cient R2 of 0.99997.

Sedimentation equilibrium experiments were con-ducted with a Beckman Optima XL-I analytical ultracen-trifuge. Rotor speeds of 20,000 and 30,000 rpm wereused at 20 �C. Samples of N-TIMP-1 (120 ml) at approxi-mately 50, 200 and 800 mM were loaded into 3 mm, six-channel cells. Absorbance at 280 nm (for 50 mM N-TIMP-1) or interference fringes (for 200 mM and 800 mM) weremeasured as a function of time and radius. The datawere analyzed using the XL-I data analysis software andOrigin 5.0 (Microcal Software, Inc.).

NMR spectroscopy

Spin-lattice (longitudinal) relaxation rate constants(R1), spin-spin (transverse) relaxation rate constants (R2),and 15N{1H} steady-state heteronuclear NOEs of thebackbone 15N nuclei were measured at 8 �C and 20 �C ona Bruker DRX500 spectrometer ®tted with an 8 mm tri-ple resonance probe with shielded Z gradient coil(Nalorac). The measurements were also performed at20 �C on a Bruker DMX750 spectrometer with 5 mm tri-ple resonance probe with shielded XYZ gradient coils.Pulse sequences for measuring R1 using inversion-recov-ery (Peng & Wagner, 1992), R2 with removal of cross-cor-relation (Kay et al., 1992), and 15N{1H} NOE (Barbatoet al., 1992) were each enhanced with pulsed ®eld gradi-ents for artifact suppression (Bax & Pochapsky, 1992)and with 3-9-19 WATERGATE for water suppression(Sklenar et al., 1993). Transverse cross-relaxation rates(Zxy) (Tjandra et al., 1996) were obtained at 8 �C,500 MHz. Zxy is the rate constant for cross-correlation orrelaxation interference between 15N-1H dipolar inter-actions and 15N chemical shift anisotropy (CSA). Delayvalues used at 500 MHz were: 16*, 32, 48*, 64, 96*, 128,and 160* ms for R2 measurements with CPMG tcp delayof 892 ms; and 20*, 80, 160*, 320, 800*, 1600, and 3000*ms for R1 inversion-recovery measurements; and 25, 40,64, and 90 ms for Zxy measurements. Asterisks indicatetime-points duplicated to allow estimation of uncertain-ties in peak heights. Delay values used at 750 MHzwere: 16*, 32, 48, 64*, 80, 96, and 112* ms for the R2

experiments; and 20*, 180, 360*, 540, 750*, 1000, 3000,and 5000 ms for the R1 experiments. In order to estimatethe conformational exchange rates of the residues experi-encing chemical exchange, additional R2 relaxation datasets with CPMG tcp delays of 392, 642, 892, and 1142 mswere recorded at 8 �C and 20 �C, respectively. A recoverydelay of 2.8 seconds was used for all R1 and R2 exper-iments mentioned above. For the 15N{1H} NOE measure-ments, two spectra, acquired with or without threeseconds of proton saturation during the recovery delay,were recorded in an interleaved manner in order to mini-mize systematic differences. The saturated experimentshad relaxation delays of ®ve seconds at 500 MHz and7.5 seconds at 750 MHz (>5 15N T1). The non-saturatedexperiments used relaxation delays of 12 seconds toensure full recovery of the 1H2O magnetization. Identicalpairs of NOE spectra were collected in triplicate at 500MHz and in duplicate at 750 MHz. The RMSD errorreported for the NOE ratios in Figure 2(a) was calculatedsuch that the squared deviations from the mean ratioIsat/Inosat for each residue were averaged over all resi-dues and then the square-root taken, similar to earlierreports (Palmer et al., 1991; Stone et al., 1993).

Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1 549

Data processing and analysis

Spectra were processed using SYBYL TRIAD version6.3 software (Tripos Inc., St. Louis, MO). Typically, a75 �-shifted squared sine bell apodization function wasapplied in both dimensions prior to zero ®lling to the®nal matrix size (2048 � 1024), Fourier transformation,and phase correction. The amide peak assignments havebeen reported (Wu et al., 1999). Individual peak heightswere measured from each spectrum for all residueswhose signals were resolved suf®ciently. Peak heightuncertainties were determined from the duplicate spectraas described (Ye et al., 1999). R1 and R2 values werederived by non-linear least-squares ®tting for the exper-imental data to an exponentially decaying function usingthe Levenberg-Marquardt algorithm implemented inKaleidagraph, version 3.08 (Synergy Software). To obtainZxy values, the experimental Icross/Iauto ratios were ®t toa tanh(tZ) function. Icross and Iauto are the intensities ofpeaks in the cross-relaxation and auto-relaxation exper-iments and t is the dephasing delay. The uncertainties inrelaxation rate constants were taken to be the standarderrors of the ®tted parameters.

Amide 15N relaxation is dominated by dipolar inter-action with its attached proton and is in¯uenced by itschemical shift anisotropy (Abragam, 1961):

R1 ��d2=4��J�oH ÿ oN� � 3J�oN

� 6J�oH � oN�� � c2J�oN� �2�

R2 ��d2=8��4J�0� � J�oH ÿ oN� � 3J�oN�� 6J�oN� � 6J�oH � oN��� �c2=6��4J�0� � 3J�oN�� � Rex �3�

NOE �1� �d2=4R1��gH=gN��6J�oH � oN�ÿ J�oH ÿ oN�� �4�

where d � m0hgNgHhrNHÿ3 i/(8p2); c � oN(sk ÿ s?)/

p3; m0

is the permeability of free space; h is Planck's constant;gH and gN are the gyromagnetic ratios of 1H and 15N,respectively; rNH � 0.102 nm is the nitrogen (hydrogenbond length; oH and oN are the Larmor frequencies of1H and 15N, respectively; and sk ÿ s? is the chemicalshift anisotropy.

The model-free calculations were performed using theModelFree 4.0 suite of programs provided by Dr ArthurPalmer (Palmer et al., 1991). The program pdbinertia wasused to estimate the ratio of the principal moments ofinertia of the solution structure of N-TIMP-1 (PDB acces-sion code 1d2b). Initial estimates of the components ofthe rotational diffusion tensor of N-TIMP-1 wereobtained from R2/R1 ratios of 70 residues, excluding resi-dues undergoing pico- to nanosecond motion or chemi-cal exchange in micro- to milliseconds (Kay et al., 1989;Palmer et al., 1991), using the program R2R1_diffusion.Representative model 22 of the high-resolution solutionstructure of N-TIMP-1 (PDB accession code 1d2b) (Wuet al., 2000) was used for such estimates.

Selections among ®ve dynamical models of increasingcomplexity of Lorentzian and Rex terms (Table 1) weremade using w2 and F-statistics to decide the simplestmodel suf®cient for the ®t, as described (Mandel et al.,1995). For assigning a model to each residue, tm and Dk/D? values were ®xed to the values initially estimated

from the R2/R1 ratios. The 15N CSA was approximatedat ÿ170 ppm (Tjandra et al., 1996), though the 15N CSAvalues of ubiquitin appear to be distributed widely(Fushman et al., 1998). The uncertainties for R2 were setto 5 % of their values instead of the smaller (2 or 3 %)values obtained from the non-linear least-square ®ts, aspreviously suggested (Volkman et al., 1998). After modelselection for each residue, a ®nal optimization was per-formed in which the overall rotational diffusion modeland the internal motional parameters for each NH vectorwere optimized simultaneously. Uncertainties in thedynamics parameters were obtained using 300 steps ofMonte Carlo simulations carried out by the ModelFreeprogram.

Reduced spectral density mapping

The spectral density function, J(o), for an N-H bondvector describes the frequency spectrum of its reorienta-tion. With the conservative assumptions that J(o)decreases monotonically with frequency, o, and hasnearly zero slope at high frequency, i.e.J(oH) � J(oH � oN) � J(oH ÿ oN), the spectral densityfunction for each amide can be approximated throughmeasurement of just the 15N R1 and R2 relaxation ratesand {1H}-15N NOE enhancements, yielding the followingspectral density expressions (Farrow et al., 1995; Peng &Wagner, 1995):

Jeff�0� � �6=�3d2 � 4c2���ÿR1=2� R2 ÿ 3s=5� �5�

J�oN� � �4=�3d2 � 4c2���R1 ÿ 7s=5� �6�

J�0:87oH� � 4s=�5d2� �7�

s � �NOEÿ 1��gN=gH�R1 �8�The constant d and the ®eld-dependent value of c arede®ned above. The reduced spectral mapping approachyields estimates of the magnitude of the spectral densityat these frequencies without assuming models of theinternal motion or models of anisotropy in the mol-ecule's rotational diffusion. Discrete values of J(o) ato � 0, oN and 0.87oH were calculated at 500 MHz and750 MHz, respectively.

Acknowledgements

The authors are most grateful to the staff of NMRFAM(Madison) who provided access to the DMX-750, to N.Murali and the staff of NHMFL (Tallahassee) who pro-vided access to the Unity� 720 for similar relaxationdata not shown, to Arthur Palmer who provided theModelFree software version 4.0, and to Michael Henzlwho provided assistance with the Beckman analyticalultracentrifuge. This work was supported by DHHSaward R01 GM57289 to S.R.V. The Bruker DRX-500 spec-trometer was funded, in part, by NSF grantCHE8908304. The Beckman XL-I analytical ultracentri-fuge was funded by NSF grant DBI9604733. NMRFAMreceives support from the NIH Biomedical TechnologyProgram (RR02301) and additional equipment fundingfrom the University of Wisconsin, NSF Academic Infra-structure Program (BIR-9214394), NIH Shared Instru-

550 Slow Dynamics of the MMP-Binding Ridge of Human TIMP-1

mentation Program (RR02781, RR08438), NSF BiologicalInstrumentation Program (DMB-8415048), and USDepartment of Agriculture. This is a contribution fromthe Missouri Agricultural Experiment Station: JournalSeries Number 13,038.

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Edited by P. E. Wright

(Received 5 May 2000; received in revised form 12 June 2000; accepted 21 June 2000)