Interdomain Interactions Support Interdomain Communicationin Human Pin1

Preparation of the I28AMutant

To make the single-sitealanine mutant I28A, we used site-directed mutagenesis by PCR, startingfrom the wild-type Pin1 construct. The mutated construct was verifiedby DNA sequencing (Genetics and Bioinformatics Core Facility at theUniversity of Notre Dame). I28A was overexpressed and isotope-labeledusing Escherichia coli BL21 (DE3) (Novagen)cells at 25 °C with 50% (v/v) D₂O M9 media with ¹⁵NH₄Cl and d-glucose (¹³C₆) as the sole nitrogen and carbon sources, respectively.Overexpression and purification followed procedures outlined in ourprevious Pin1 work.¹ The final sample wasconcentrated and dialyzed against the NMR buffer: 30 mM imidazole-d₄ (pH 6.6), 5 mM DTT-d₁₀, 30 mM NaCl, 0.03% NaN₃, and 90% H₂O/10% D₂O. Folding was confirmed by comparing the two-dimensional(2-D) ¹⁵N–¹H heteronuclear single quantumcorrelation (HSQC) spectra of I28A with wild-type Pin1.

Far-UV CircularDichroism (CD) Spectroscopy

CD measurementswere performed in 20 mM NaH₂PO₄, pH 7.5 on aJasco J-815 spectropolarimeter. Far-UV CD spectral acquisitions useda 1 mm path length cuvette with protein concentrations ranging between2 and 10 μM. Thermal denaturation was monitored at 200 nm overa temperature range of 20–80 °C with 1 min thermal equilibrationsfor each 1 °C step.

NMR Resonance Assignments and Chemical ShiftPerturbations

We recorded NMR spectra at 295 K using BrukerAvance 700 MHz (16.4T) and 800 MHz (18.8 T) spectrometers equipped with cryogenicallycooled probes. I28A backbone assignments (¹H^N, ¹⁵N, ¹³Cα, and ¹³Cβ)were confirmed using three-dimensional (3-D) HNCACB,²⁵ HNCOCACB,²⁶ and 2-D ¹H–¹⁵N HSQC²⁷ pulse schemes.Side-chain aliphatic ¹³C and ¹H resonances wereassigned by comparisons with the corresponding wild-type Pin1 spectra.Fourier transformation and NMR resonance assignment used Topspin 1.3and 2.1 (Bruker Biospin, Inc.) and Sparky (T. D. Goddard and D. G.Kneller, SPARKY 3, University of California, San Francisco). The ¹⁵N–¹H chemical shift perturbations of theprotein in state A relative to state B were defined as1where Δδ_N^A–B = δ_N^A – δ_N^B and Δδ_H^A–B = δ_H^A – δ_H^B. For assessingmutation effects, A was the mutant, whereas B was wild-type Pin1.For assessing ligand-binding effects, A was the protein–ligandcomplex, whereas B was the apo protein. Backbone ¹³Cα/βshift changes between apo I28A and apo WT Pin1 were determined fromcomparing their HNCACB/HNCOCACB spectra.

K_d Values from Chemical Shift Perturbations

For bindingstudies, the chemical shift perturbations were interpretedin terms of a simple equilibrium2where P, L, and PL are the free protein,freeligand, and protein–ligand complex, respectively. We fittedthe chemical shift perturbation versus the ratio of total ligand tototal protein (L_T/P_T) to3Equation 3 assumes fastbinding exchange on the chemical shift time scale. The adjustableparameters were the equilibrium dissociation constant, K_d, and the maximum chemical shift perturbation at saturation,Δδ_NH,max. Uncertainties in the fitted parameterswere estimated by jack-knife simulations.

Measurements of Cis–TransIsomerase Activity

We used two methods to measure cis–transisomerase activity.The first method was the standard chromogenic coupled-assay of Kofronet al.²⁸ The chromogenic substrate, suc-AEPF-pNAwas purchased from Sigma. The assay procedures were the same asin our previous Pin1 study.¹ The secondmethod was 2-D ¹H–¹H exchange spectroscopy(EXSY), using a NOESY-based pulse scheme.²⁹ The substrate was a ten-residue phosphothreonine peptide, EQPLpTPVTDL(Anaspec), which is an established proxy for the Pin1 target sitein the mitotic phosphatases Cdc25C.^14,16 Experimentswere carried out at 18.8T (800 MHz ¹H Larmor frequency),295 K. The mixing times for exchange were 1, 20, 30, 40, 50, 60, 80,100, and 200 (×2) ms. Samples consisted of 50 μM freshprotein (wild-type Pin1, I28A, or the isolated PPIase domain) in thepresence of 2 mM Cdc25C phosphopeptide substrate. Exchange rate constants, k_EXSY, were estimated by fitting the ratios ofthe trans-to-cis exchange cross-peaks over the trans diagonal peaksas function of the mixing time t_mix, tothe two-state expression^29,304The two adjustable parameters were k_TC and k_CT and k_EXSY = k_TC + k_CT. The exchange cross-peaks were assigned bycomparison to 2-D ¹H–¹H total correlationspectroscopy (TOCSY)^31,32 and rotating-frame nuclear Overhausereffect correlation spectroscopy (ROESY)³³ spectra. In samples containing just 2 mM Cdc25C phosphopeptide (noprotein), the EXSY cross-peaks were absent because the exchange wasbeyond the limit of detection (too slow).

NMR Spin Relaxation Experimentsand Analysis

All backbone R₁(¹⁵N), R₂(¹⁵N), and steady-state ¹H^N–¹⁵N NOE (ssNOE) values weremeasured at 16.4 T (700 MHz ¹H Larmor frequency) usingpreviously established 2-D ¹⁵N–¹H pulseschemes,^34,35 at 295 K. The delays for R₁(¹⁵N) were t_relax = 200.5 (2×), 411.4,633.0, 833.4, 1044.4, 1266.0, 1677.4, and 2099.4 ms. The delays for R₂(¹⁵N) were multiples of the basicCPMG echo block (8.48 ms) that included pulses and delays to removeunwanted cross-correlated relaxation effects;^36,37 this resulted in t_relax = 17 (2×),25.4, 33.9, 42.4, 50.9, 59.4, 76.3, and 93.3 ms. For the ssNOE, werecorded interleaved pairs of spectra in which ¹H saturationof 5 s was applied alternately.

Deuterium relaxation rates R_1ρ (²D) and R₁ (²D) for CH₂D methyl groups were measuredwith established 2-D ¹³C_methyl–¹H_methyl pulse schemes^38,39 at 16.4 and18.8 T (700.13 and 800.13 MHz ¹H Larmor frequencies), 295K. ²D hard-pulses were applied at 1.78 kHz, while the spin-lockswere applied at reduced strengths of 1.2 kHz. The delays for R_1ρ (²D) were t_relax = 0.5 (2×), 1.5, 2, 3, 4, 6.5, and 8 ms at 700 and800 MHz. The R₁ (²D) delayshad t_relax = 0.05 (2×), 10, 15, 21,31, 42, and 50 ms at both 700 and 800 MHz.

All relaxation analysisused in-house software written in C programminglanguage. Cross-peak intensities were measured by integrating alongf₂ (¹H dimension) through the cross-peak maximain f₁ (¹³C or ¹⁵N), which gave foreach resonance a file of cross-peak intensities “I” versus relaxation delay “t_relax”. The I(t_relax) versus t_relax fileswere fit to two-parameter single-exponential decay functions, I(t_relax) = A exp{−Rt_relax}, where R is the desired relaxation rate constant. Statistical errorswere estimated using Monte Carlo methods with duplicate spectra furnishingthe integral uncertainties.

To describe the backbone NH bondmotions, we determined reducedspectral density values⁴⁰⁻⁴², J_eff^NH(0), J^NH(ω_N), and ⟨J^NH(ω_H)⟩, using the relations⁴²5The σ_NH wasextracted from the measured ssNOE and R₁(N) via6The C and D constants in eq 5 reflect the ¹⁵N chemical-shift-anisotropy and ¹⁵N–¹H dipolar relaxation mechanisms, respectively, and were C = Δ²ω_N²/3 and D = ℏ²γ_H²γ_N²/⟨r_HN⁶⟩ (cgsunits).

The deuterium (spin-1) relaxation rates are dominatedby the quadrupolarrelaxation mechanism, resulting in rate constant expressions,⁴³78J^CD(ω) isthe spectral density function that reports on the reorientationalmotions of the ¹³C–²D bond vectors withrespect to the external magnetic field, B₀. We used a quadrupolar coupling constant Q_CC = (e²qQ/ℏ)= 2π*167 kHz. To extract dynamics parameters from the relaxation rates,we used eqs 7 and 8 tofit the R_1ρ(²D) and R₁(²D) rates to an analytical J^CD(ω) function given by the Lipari–Szaboformalism^39,44,459Equation 9 assumes thatthe “twirling” motions of the C–D bond vectorsabout the methyl symmetry axis are completely averaged out (extreme-narrowing),resulting in the factor of 1/9. Hence, the site-specific motions ofthe ¹³C–²D bond vectors are actuallythose of the corresponding methyl symmetry axes, with amplitudes givenby the order parameter S_AXIS². The parameter τ satisfies 1/τ= 1/τ_m + 1/τ_e, where τ_m is the global correlation time for overall tumbling, andτ_e is a site-specific correlation time related tomotions underlying S_AXIS².

For the overall tumbling correlationtime τ_m,we used the Levenberg–Marquardt algorithm⁴⁶ and fit the ratios R₂(¹⁵N)/R₁(¹⁵N) of eachdomain (WW and PPIase) to get domain-specific τ_m values.The fit included only ratios within one-standard deviation of theraw mean. For each domain, we kept its τ_m fixed andused the Levenberg–Marquardt algorithm to fit S_AXIS² andτ_e for the individualmethyls.⁴⁶ Errors in S_AXIS² andτ_e were estimatedusing Monte Carlo simulations based on the estimated uncertaintiesin the experimental rate constants. Methyls excluded from this fittingbecause of resonance overlap included V22Cγ₂, A31Cβ,L61Cδ₂, L88Cδ₂, L106Cδ₁, L122Cδ₂, T152Cγ₂, andT162Cγ₂. Further details are in our previous studiesof Pin1 side chain-dynamics.^1,2

Backbone ChemicalShift Perturbations

The goal of theI28A substitution was to weaken interdomain contact within apo Pin1while maintaining the overall folds of both domains. Comparisons of2-D ¹⁵N–¹H HSQC spectra for U–¹⁵N/¹³C, 50% ²D I28A and wild-type Pin1(WT) indicated we had achieved our goal (Figure 2). The majority of HSQC cross-peaks of I28A were in the same positionsas those of WT, indicating unchanged NH chemical shifts. We also compared ¹³Cα/β chemical shifts for I28A versus WT (FigureS1 of the Supporting Information) becausethese shifts are sensitive probes to local torsion angles and theirfluctuations.⁴⁷⁻⁴⁹ The majority of residues showed small (<0.3 ppm)or no ¹³Cα/β shift perturbations. Thus, togetherthe ¹⁵N and ¹³Cα/β chemical shiftssuggested preservation of the overall WT fold, albeit, with localstructural perturbations. Far-UV CD spectra corroborated the preservationof the overall fold (Figure S2 of the SupportingInformation). In particular, wave scans of I28A and WT at 20°C were essentially identical. Moreover, their thermal meltsfollowed by far-UV CD at 200 nm were identical (T_m,I28A = 62.1 ± 0.2 °C versus T_m,WT = 62.5 ± 0.2 °C).

Figure 2

Comparison of 2-D ¹⁵N–¹H HSQC NMRspectra for I28A (red cross-peaks) versus wild-type Pin1 (blue cross-peaks)at 16.4 T, 295 K.

We focused mainly onthe 2-D ¹⁵N–¹H spectra to determine theeffect of the I28A mutation on interdomaincontact. Although the 2-D ¹⁵N–¹H I28Aand WT spectra were overall quite similar, there were important localchemical shift perturbations. We quantified these perturbations, Δδ_NH^I28A–WT =δ^I28A – δ^WT, using eq 1 (Materials and Methods).As expected, I28A showed prominent Δδ_NH^I28A–WT for its neighboringresidues N16 and T29 (Figure 3, upper panel),but there were also perturbations in the PPIase domain. Some wereunexpectedly long-ranged, occurring on the far side of the PPIasedomain (PPIase catalytic loop, L60, K77, K82, and A107). These ¹⁵N–¹H shift perturbations were corroboratedby similar long-range ¹³Cα shift perturbations (FigureS1 of the Supporting Information). Specifically,beyond the expected ¹³Cα shift perturbations at WWdomain residues at and flanking the mutation site, there were alsoa handful of significant ¹³Cα perturbations in thePPIase domain, including (i) R54 at the C-terminal end of the interdomainlinker, (ii) S67, K77 in the catalytic loop, (iii) S105 and F110 inα2, and (iv) I158 at the C-terminus. Most revealing, however,were the ¹⁵N–¹H shift perturbations atthe PPIase residues at the C-terminal end of α4 including S138–R142.These PPIase residues lay across the domain interface from the I28Amutation site and its host Loop II in the WW domain (see Figure 1). Moreover, these perturbations matched those weobserved for the isolated WT PPIase domain, relative to full-lengthPin1, Δδ_NH^PPIase–WT (Figure 3, lower panel).In effect, the pattern of chemical shift perturbations in apo I28Amatched those caused by the deletion of the WW domain. This was strongevidence that the I28A mutation had weakened the interdomain contactof apo Pin1.

Figure 3

Backbone ¹⁵N–¹H chemicalshift perturbationsfor apo protein constructs. Top panel: perturbations Δδ_NH^I28A–WT forapo I28A relative to apo WT. Bottom panel: Perturbations Δδ_NH^PPIase–WT caused by deletion of the WW domain (i.e., isolated apo PPIase domainrelative to apo full-length WT). Green spheres are common chemicalshift perturbations >0.05 ppm at the domain interface; red spheresare all other perturbations >0.05 ppm.

Additional evidence for weakened interdomain contact camefromcomparing the chemical shift perturbations caused by substrate binding(i.e., Δδ_NH^{COMPLEX–APO}) in I28A and WT Pin1. Specifically, previousstudies showed that WT binding of the phosphopeptide substrate EQPLpTPVTDL,a proxy for the Pin1 target Cdc25C phosphatase from Xenopus laevis,^14,16 caused significantchemical shift perturbations at S138–R142^1,21 (Figure 4, lower panel). These WT chemical shift perturbationswere the signature response indicating increased interdomain contactstimulated by substrate binding.²¹ By contrast,adding saturating amounts of Cdc25C substrate to I28A failed to showthis response (Figure 4, upper panel). Thisfailure of response in I28A is consistent with its weakened interdomaincontact.

Figure 4

Backbone ¹⁵N–¹H chemical shift perturbationsof I28A (top) and WT (bottom) caused by adding saturating amountsof Cdc25C phosphopeptide substrate, EQPLpTPVTDL. Green spheres highlightdomain interface shift perturbations >0.05 ppm for WT, which aremostlyabsent in I28A. Red spheres indicate all other perturbations >0.05ppm. Light aquamarine and magenta shading indicate the PPIase andWW domains, respectively.

Cis–Trans Isomerase Activity

We investigatedthe effect of the I28A mutation on cis–trans isomerase activityusing two methods. We first measured activity via the standard chromophoriccoupled assay of Kofron et al.,²⁸ whichuses the substrate suc-AEPF-pNA. We found that I28A showed an ∼36%reduction of the specificity constant, k_cat./K_M, relative to WT (I28A, 2724 ±140 mM^–1 s^–1; WT, 4250 ±213 mM^–1 s^–1). This result echoesthat of the isolated WT PPIase domain, which also showed a slightdecrease of k_cat./K_M relative to WT.¹⁶

We alsomeasured I28A isomerase activity using 2-D ¹H–¹H NMR exchange spectroscopy (EXSY),²⁹ which used the Cdc25C phosphopeptide substrate mentioned above.EXSY spectra produced cross-peaks corresponding to pT5 methyl protonsexchanging between the cis versus trans chemical shifts. For bothI28A and WT, we fitted the time course of these exchange cross-peaksto the two-state exchange expression in eq 4 (Materials and Methods) to get a net exchangerate constant, k_EXSY = k_TC + k_CT.³⁰ It is important to note that k_TC and k_CT are the apparent rate constantsfor trans-to-cis and cis-to-trans exchange and are functions of K_M and k_cat. forthe corresponding trans-to-cis and cis-to-trans isomerization processes.⁵⁰ I28A showed increased k_EXSY relative to WT (i.e., k_EXSY(I28A) = k_CT + k_TC = 73 ± 2 s^–1 versus k_EXSY(WT) = k_CT + k_TC = 31.3 ± 0.5 s^–1) (Figure S3of the Supporting Information). This increaseechoed our previous observation of increased k_EXSY for isolated WT PPIase domain relative to full-lengthWT Pin1.⁵¹

Thus, the I28A mutationaltered the cis–trans isomerizationactivity, despite the remote location of I28 from the PPIase activesite. The sense of alteration matched that observed when going fromfull-length Pin1 to the isolated PPIase domain. In particular, bothI28A and the isolated PPIase domain show the same trend of slightlyincreased k_EXSY (2-D EXSY) and slightlydecreased k_cat./K_M (chromogenic assay) relative to WT.

Effect of I28A on SubstrateBinding Affinity

To assessthe mutation’s impact on substrate binding, we titrated I28Awith the Cdc25C phosphopeptide and fitted the resulting NH chemicalshift perturbations of resolved NH cross-peaks to eq 3 (Materials and Methods). This gavesite-specific estimates for binding affinity (i.e., theequilibrium dissociation constant K_d).The values are listed in Table 1, and the correspondingisotherm fits are in Figure S4 of the SupportingInformation. Compared with WT, I28A showed weaker binding affinity(i.e., K_d^I28A > K_d^WT) in both domains. The affinitydecrease varied, with the ratios K_d^I28A > K_d^WT values rangingfrom ∼5 to ∼10. The spread partly reflected the lowsignal-to-noise of some cross-peaks; nevertheless, the general trendof an affinity decrease was unambiguous. The decreased binding affinitywas striking, given that neither I28 itself nor its host loop directlycontact phosphopeptide substrate.^13,14

Backbone Mobility of apo I28A

We previously characterizedthe changes in WT Pin1 backbone and side chain flexibility causedby binding the Cdc25C phosphopeptide substrate.¹ It was therefore of interest to see whether I28A wouldhave similar responses, in light of its altered substrate bindingaffinity and cis–trans isomerase activity.

We first characterizedthe backbone flexibility of apo I28A, by measuring backbone amide ¹⁵N relaxation parameters, R₁, R₂, and steady-state ¹⁵N–¹H NOE at 16.4 T, 295 K. We analyzed the relaxation data usinga reduced NH spectral density mapping procedure⁴² that produced for each NH a value for J_eff^NH(0), alocal mobility parameter. J_eff^NH(0) is the zero-frequency value forthe effective NH spectral density function J_eff^NH(ω) thatdescribes the reorientational motions of the NH bonds relative tothe external magnetic field, B₀. For a rigid,isotropically tumbling molecule, J_eff^NH(0) should be uniform acrossall NH bonds. NH bonds with outlying J_eff^NH(0) values highlightsites of internal motion. In particular, high J_eff^NH(0) outliersindicate dynamic processes modulating the ¹⁵N chemicalshift on the microsecond to millisecond time-scale, whereas low J_eff^NH(0) outliers reflect large amplitude, internal motions that reorientthe NH bond on the subnanosecond time scale.⁴² Comparing J_eff^NH(0) values for apo I28A with those we hadpreviously obtained for apo WT¹ allowedus to assess the mutation’s affect on intrinsic backbone dynamics.

We found that the overall profile of J_eff^NH(0) versus thesequence for apo I28A was similar to apo WT, in that the J_eff^NH(0) valueshad partitioned into two distinct clusters, corresponding to the WWand PPIase domains. This clustering indicated that the overall moleculartumbling of I28A, like that of WT, could be approximated as two domainstumbling in quasi-independent manner with domain-specific correlationtimes, τ_m,WW and τ_m,PPI.

Nevertheless,the apo I28A showed important local differences in J_eff^NH(0) comparedwith apo WT. To highlight these differences, we usedthe site-specific ratio J_eff^NH,I28A(0)/J_eff^NH,WT(0). Providedthe mutation does not affect internal mobility, this ratio shouldbe the same for all NHs within a given domain. Thus, those NH bondsthat display outlying ratios represent sites experiencing mutation-inducedchanges in internal motion. We screened for such outliers by identifying J_eff^NH,I28A(0)/J_eff^NH,WT(0) ratios beyond one standard deviationfrom the trimmed mean value for the WW or PPIase domain, as appropriate.Figure 5 shows the results; the spheres/barscolored blue and red indicate low and high outliers, respectively.The most prominent high outliers were WW domain residues with J_eff^NH,I28A(0)/J_eff^NH,WT(0) > 1. These are residues whose NHbondsexperience microsecond to millisecond exchange dynamics in I28A thatare lacking in WT. These residues include N26, T29, and N30, whichlie within or adjacent to Loop II, and bracket I28. Surprisingly,they also included WW domain residues outside Loop II, notably K13and R14 in the β strand, β1′. Possible reasonsfor these surprising β1′ changes are in the Discussion. Low outliers, J_eff^NH,I28A(0)/J_eff^NH,WT(0) < 1, indicating enhanced subnanosecond mobility of apo I28Aversus apo WT, were distal from the mutation site and occurred inthe linker, the PPIase domain catalytic pocket, and the flexible PPIaseloop (H64–R80) “capping” the catalytic pocket.

Figure 5

(A) Pin1colored to highlight changes in backbone NH dynamics inapo I28A compared with apo WT. Ribbons colored aquamarine, magenta,and yellow indicate the PPIase, WW,and PPIase catalytic site regions, respectively. The red/blue spheresare NHs with outlying values of J_eff^I28A(0)/J_eff^WT(0) (ratio values>1 standard deviation from the domain-specific trimmed mean). Redspheres highlight NH sites showing enhanced exchange dynamics, whereasblue spheres are sites with enhanced subnanosecond flexibility. (B)Bar graph showing the data underlying panel A. The bars are deviationsof NH J_eff^I28A(0)/J_eff^WT(0) from the domain-specifictrimmed means. Red/blue bars correspond to the red/blue spheres in(A).

Backbone Mobility of I28Ain the Presence of Substrate

We carried out the same backbone ¹⁵N relaxation analysisdescribed above for I28A in the presence of saturating amounts ofCdc25C phosphopeptide substrate. To highlight changes in internalmotion caused by substrate binding, we used the ratio J_eff^NH,COMPLEX(0)/J_eff^NH,APO(0), where the J_eff^NH,APO(0) valueswere those from the apo measurements described above. If substratebinding simply alters the domain rotational correlation time (e.g.,τ_m,WW or τ_m,PPI), then the J_eff^NH,COMPLEX(0)/J_eff^NH,APO(0) ratios should be the same across allNHs within a given domain. Thus, those NH bonds that show outlyingratios represent bonds whose local dynamics have changed upon substratebinding. Ratios were identified as outliers if they deviated fromthe trimmed domain average by more than one standard deviation. Wetherefore compared the number and location of J_eff^NH,COMPLEX(0)/J_eff^NH,APO(0) outliers for I28A and WT, as a means to compare their dynamicresponses to substrate binding.

Comparing these outliers revealedthat Cdc25C substrate binding caused backbone dynamic changes in I28Athat were absent in WT. Specifically, Figure 6 shows the outliers for both WT and I28A; the spheres/bars coloredblue and red indicate low and high deviations, respectively. I28Ahad many outliers in the WW domain that were absent in WT. These outliersreflect the quenching of the aforementioned microsecond to millisecondexchange dynamics of apo I28A in Loop II in apo I28A, upon substratebinding. Interestingly, I28A also showed outliers at S58, V62, andC113, which are part of the substrate proline binding pockets withinthe PPIase domain. For these three residues, the ratio J_eff^NH,COMPLEX(0)/J_eff^NH,APO(0) became smaller, with the denominator J_eff^NH,APO(0) value close to the domain average; this indicated increased subnanosecondflexibility upon substrate Cdc25 phosphopeptide binding. Such a responsewas utterly lacking in WT. Thus, the I28A mutation in the WW domainchanged the dynamic response of residues in the distal PPIase domainto substrate binding. Other sites outside the substrate binding pocketshowing distinctly different dynamic response for I28A included K82and E83, at the juncture between the catalytic loop and the long helixα1.

Figure 6

Site-specific changes in NH backbone dynamics caused by Cdc25Csubstrate for both WT (top panel (A)) and I28A (bottom panel (B)).All structures are from PDB id 1PIN. Ribbon colors of aquamarine, magenta,and yellow ribbon indicate the PPIase, WW, and PPIase catalytic siteregions, respectively. The red/blue spheres are NHs with outlyingvalues of J_eff^NH,COMPLEX(0)/J_eff^NH,APO(0) (ratios>1 standard deviation from the domain-specific trimmed mean) andthusindicate binding-induced changes in local mobility. The red/blue barsin the bar graphs correspond to the red/blue spheres in the structures.In the bottom panel (B), S58, V62, and C113 are PPIase catalytic siteresidues that show increased subnanosecond mobility upon substratebinding for I28A but not for WT. K82 and E83, at the juncture betweenthe catalytic loop and the long helix α1, also show dynamicchanges not found in the WT.

Subnanosecond Side-Chain Mobility in apo I28A

We theninvestigated the impact of the I28A mutation on side-chain flexibility,specifically, the subnanosecond reorientational motions of methylsymmetry axes with respect to the magnetic field, B₀. This involved measuring ²D R₁ and R_1ρ rate constantsfor all methyl CH₂D isotopomers in U–¹⁵N, ¹³C, 50% ²D enriched I28A at 16.4 and 18.8T, 295 K. We analyzed the resulting rates using the familiar Lipari–Szaboformalism.⁴⁴ This produced two methyl-specificdynamics parameters: S_AXIS² and τ_e, per eq 9 (Materials and Methods). S_AXIS² is a pure number that measures theamplitude of reorientational dynamics of a methyl symmetry axis, dueto subnanosecond internal motions. S_AXIS² rangesfrom 0 to 1: a value of 0 correspondsto unrestricted internal motion, whereas a value of 1 correspondsto no internal motion (rigid symmetry axis). The τ_e parameter is an effective correlation time that estimates the rapidityof the internal orientational dynamics but also depends on the amplitudeof motion.⁴⁴

Fitting S_AXIS² andτ_e relies onprior characterization of the overall molecular tumbling. The backboneNH reduced spectral density analysis above justified approximatingthe overall I28A tumbling in terms of domain-specific rotational correlationtimes. Accordingly, we determined τ_m,WW and τ_m,PPIase, using the R₂(¹⁵N)/R₁(¹⁵N) ratios³⁵ of only those backbone NHs with J_eff(0) within 1 standard deviation of the trimmed mean.For apo I28A, we found τ_m,WW = 7.8 ± 0.01 ns/r,τ_m,PPIase = 12.0 ± 0.01 ns/r, and for the Cdc25Ccomplex τ_m,WW = 7.5 ± 0.01 ns/r and τ_m,PPIase = 11.4 ± 0.01 ns/r. With the domain-specificoverall tumbling times set, we were able to fit the site-specificside-chain internal motion parameters, S_AXIS² and τ_e. We then compared S_AXIS² from apo I28A withthose we previously determined for apo WT,¹ by evaluating the differences ΔS_AXIS,APO² = S_{AXIS,APO:I28A}² – S_AXIS,APO:WT².

Remarkably, despite the fact that the I28A mutation was in theWW domain, it produced widespread changes in intrinsic side-chainflexibility (ΔS_AXIS,APO²) in both domains. In particular, Figure 7, top panel, maps these differences onto the 1PIN crystal structure.Red spheres and positive bars indicate ΔS_AXIS,APO² >0,and pinpoint methyl axes for which I28A was more rigid than for WT.Blue spheres and negative bars indicate the opposite trend. In theWW domain, we saw greater I28A flexibility at L7Cδ₁; notably L7 has key hydrophobic interactions with W11, one of thetwo conserved tryptophans of the WW domain. In the PPIase domain,we observed both mobility increases and decreases. Sites where I28Aloosened relative to WT (ΔS_AXIS,APO² < 0) included (i)V62Cγ₂ in the PPIase β4 strand near the PPIaseactive site, (ii) T81Cγ₂, A85Cβ, and I89Cδ₁ in the long PPIase α1 helix, and (iii) A116Cβin α3 helix. Sites where I28A stiffened compared with WT (ΔS_AXIS,APO² > 0) included (i) V55Cγ₂, L60Cδ₁, L60Cδ₂, and L61Cδ₁ inthe PPIase active site, (ii) I93Cδ₁ and I96Cδ₁ in the long α1 helix, (iii) A140Cβ and L141Cδ₂ between α4 and β3, (iv) V150Cγ₂ in β3, and (v) I159Cγ₂ in β4. Interestingly,the last three locales (A140Cβ, L141Cδ₂, V150Cγ₂, and I159Cγ₂) are all near the domain interface.

Figure 7

Changesin methyl side-chain order parameters S_AXIS² fromdeuterium spin relaxation. Top panel: apo states for WT versus I28A,ΔS_AXIS,APO² = S_{AXIS,APO:I28A}² – S_AXIS,APO:WT².Middle panel: I28A complexed with Cdc25C phosphopeptide versus itsapo state, ΔS_AXIS,BINDING² = S_{AXIS,CMP:I28A}² – S_{AXIS,APO:I28A}². Bottom panel: Cdc25C phosphopeptidecomplexed states for WT versus I28A, ΔS_AXIS,COMPLEX² = S_AXIS,CMP:WT² – S_{AXIS,CMP:I28A}². The bars denote S_AXIS² with magnitudesgreater than or equal to twice (purple) or once (hatched) the estimatedstatistical errors. The structures to the right of each bar graphshows colored spheres corresponding to the methyls changes highlightedin the bar graphs. The red and blue spheres indicate sites of ΔS_AXIS² > 0 (more rigid) and ΔS_AXIS² < 0(more flexible), respectively. Bottom structure: residues coloredto contrast the pattern of side-chain flexibility loss in I28A uponCdc25C substrate binding, with that of the previously defined WT conduit.¹ Specifically, the red and deep salmon residuestrace the original WT conduit. The red residues are those that loseside-chain flexibility only in WT (i.e., not I28A). The deep salmonresidues lose side-chain flexibility in both WT and I28A. The greenresidues are those that lose side-chain flexibility only in I28A.The red and green residues thus highlight the departure of I28A fromthe previously defined WT conduit.

Subnanosecond Side Chain Mobility in I28A in the Presence ofSubstrate

We investigated Cdc25C substrate binding affectedI28A side-chain mobility by evaluating ΔS_AXIS,BINDING² = S_{AXIS,CMP:I28A}² – S_{AXIS,APO:I28A}². The ΔS_{AXIS,CMP:I28A}² values were from I28A in the presenceof saturating amounts of Cdc25C substrate, whereas the ΔS_{AXIS,APO:I28A}² values were from the apo studies describedabove. Positive and negative ΔS_AXIS,BINDING² indicate a loss or gain of side-chain flexibility, respectively,upon Cdc25 binding. The middle panel of Figure 7 maps these differences onto the structure. Losses of flexibility(ΔS_AXIS,BINDING² > 0) occurred at the N-terminusofthe WW domain L7Cδ₁, the PPIase domain active siteV62Cγ₂, T81Cγ₂, and A85Cβ,I89Cδ₁, L106Cδ₂, A118Cβ, L122Cδ₁, M146Cε, and V150Cγ₂. Gains in flexibility(ΔS_AXIS,BINDING² < 0) occurred at the flexible linker(A53Cβ), the PPIase domain active site L60Cδ₁ and L60Cδ₂, the domain interface L141Cδ₂, and the C-terminal L160Cδ₂. Thus, manysites that showed intrinsically different side chain mobility fromWT (ΔS_AXIS,APO²) also underwent changes in S_axis² upon substrate binding.

We wanted to compare how thebinding-induced changes in side chainflexibility for I28A compared with what we had already documentedfor WT. To this end, we compared order parameters from the two Cdc25Ccomplexes, WT/Cdc25C and I28A/Cdc25C, by evaluating the differenceΔS_AXIS,CMP² = S_AXIS,CMP:WT² –S_{AXIS,CMP:I28A}². Figure 7,bottom panel, reveals different responses in both domains. Generally,the I28A/Cdc25 complex was stiffer than the WT/Cdc25 complex. TheI28A complex showed greater rigidity than the WT complex (ΔS_AXIS,CMP² < 0) at L7Cδ₁ in the WW domain, I89Cδ₁ (PPIase α1, domain interface), L106Cδ₂ (PPIase α2), L122Cδ₁ (PPIase active site),and V150Cγ₁ V150Cγ₂, I158Cδ₁ and I159Cδ₁ (PPIase β3 and β4,adjacent to residues comprising the domain interface). On the otherhand, the WT complex showed greater rigidity than the I28A complex(ΔS_AXIS,CMP² > 0) at L7Cδ₂ (WW domain),A53Cβ (linker), and L60Cδ₁ (PPIase β1active site), and L141Cδ₂ (domain interface).

A particularly striking difference in side chain dynamic responseoccurred at L60–L61–V62, a conserved hydrophobic clusterwithin the PPIase active site. Specifically, our previous dynamicsstudies of WT showed a loss of side-chain flexibility for this conservedcluster.^1,2 By contrast, the response of I28A upon substratebinding was an increase in flexibility at L60 (Figure 7, middle panel). This flexibility increase alsoappeared in the subnanosecond backbone flexibility at these sites(Figure 5). The deviant dynamic response inthe I28A PPIase active site, for both side chain and backbone, isnoteworthy given that its isomerase activity (k_cat./K_M from the chromogenic assay,and k_EXSY from 2-D NMR) differs from WT.

Finally, the structure (PDB id 1PIN) in lower panel (B) of Figure 7 further highlights how the distribution of side-chainflexibility loss in I28A caused by Cdc25C substrate binding deviatesfrom the conduit response we first observed for the WT.¹ Specifically, the coloring denotes residues thatlose side-chain flexibility (i) only in WT (red), (ii) in both WTand I28A (deep salmon) (iii), and only in I28A (green). The red anddeep salmon residues trace the original WT conduit.

I28A Weakens Intrinsic Interdomain Contact

As our ¹⁵N–¹H NMR chemical shift perturbationsandfar-UV CD data indicate, the I28A mutation indeed weakens interdomaincontact, while preserving the overall structure of WT Pin1 (Figures 2–4, Figure S1 of the Supporting Information). The ¹⁵N–¹H shift perturbations depict an interdomain contact regionconsistent with the region depicted in the 1PIN crystal structure²² and include H27–N30 of the WW domain (Loop II) andof the PPIase domain residues S138–R142 (C-terminal residuesof α4).

The backbone ¹⁵N dynamics study ofI28A provides further support for weakened interdomain contact. LoopII (H27–N30) in I28A exhibits greater microsecond to millisecondmobility than Loop II in WT, as evidenced by elevated ratios J_eff^NH,I28A(0)/J_eff^NH,WT(0) in Figure 5.These elevated ratios indicate enhanced amide proton exchange, conformationalexchange, or both. Greater Loop II flexibility in I28A would be consistentwith its lowered commitment from Loop II to interdomain contact.

I28A Shows Isomerase Activity Consistent with Weakened InterdomainContact

The weakened interdomain contact in I28A coincideswith changes in isomerase activity. Notably, the changes of I28A relativeto WT are similar to those displayed by the isolated PPIase domain.Specifically, both I28A and the isolated PPIase domain show the sametrend of slightly increased k_EXSY (2-DEXSY assay) and slightly decreased k_cat./K_M (chromogenic assay) relative to WT.Thus, I28A mutation changes the PPIase activity in a direction thatis diagnostic of lost communication with the WW domain. These resultssuggest that interdomain contact has a functional significance, inthat it can fine-tune PPIase activity.

This fine-tuning is intriguingbecause I28 and the other residues supporting interdomain contact(e.g., H27–N30 in the WW domain Loop II and S138–R142in the PPIase domain) do not directly contact substrate. Rather, theyare spatially removed from those regions that do, namely, the PPIasedomain catalytic site and the WW domain substrate binding Loop I (S16–R21)(Figure 1). This physical separation raisesthe question of how changes in the interdomain contact, either itsweakening or elimination, could alter the activity of the distal PPIasecatalytic site.

Interdomain Contact Affects the PPIase DomainProperties

The standard explanation emphasizes the WW domain’srole asan independent binding module.^9,19 Its proposed influenceon the PPIase activity, modulating the local substrate concentration,simply reflects its proximity to the PPIase domain. Interestingly,there appears to be no consensus on what this modulation is: boththe enhancement and the depletion of local substrate concentrationhave been suggested.^11,18,52 Mutating the WW domain may compromise its ability to bind substrate,and hence, its ability to modulate local substrate concentration.Two features of this standard explanation are noteworthy. First, itdoes not explicitly invoke domain contact but merely domain proximity.Second, the view of the WW domain as an independent module impliesthat a WW domain mutation may perturb local conformation and flexibilitywithin the WW domain but not within the PPIase domain.

Our resultsfrom I28A suggest a more complex interdomain relationship. Certainly,the I28A mutation does perturb the WW domain, as evidenced by reducedCdc25C binding affinity and altered Loop II mobility (Figure 5). But in contrast to the implications of the standardexplanation, the WW domain mutation also impacts the intrinsic propertiesof the PPIase domain. First, comparisons of the apo I28A versus apoWT backbone ¹⁵N–¹H chemical shifts showperturbations in both domains, including PPIase residues far removedfrom the PPIase domain interface, such as those of the PPIase domaincatalytic loop (Figure 3). Similar remote ¹³Cα chemical shift perturbations corroborate these remote ¹⁵N–¹H perturbations (Figure S1 of the Supporting Information). Second, we found weakerCdc25 phosphopeptide substrate binding affinities (higher K_d = C⁰ exp(ΔG^PPIase,0/k_BT)) for both domains (Table 1 andFigure S4 of the Supporting Information). This suggests that the I28A WW domain mutation makes the freeenergy difference between the complexed and apo states, ΔG^PPIase,0 = G_COMPLEX^PPIase – G_APO^PPIase,less negative. Finally, the I28A mutation causes widespread changesin the intrinsic (apo state) backbone and side-chain flexibility ofthe protein, beyond the domain interface. Figure 5 and the top panel of Figure 7 showdistal changes in backbone and side-chain flexibility, respectively,at functional sites of the PPIase domain. In particular, the top panelof Figure 7 shows complementary changes inside-chain flexibility at residues within the PPIase catalytic site(V55Cγ₂, L60Cδ₁, L60Cδ₂, and L61Cδ₁). Considering the above points,the view of the WW domain as an independent binding module would appearincomplete. Rather, our data suggest that the internal propertiesof the PPIase domain are sensitive to WW domain contact and to mutationssuch as I28A that weaken that contact.

Interdomain Contact Tunesthe Dynamic Response of Pin1 to Binding

Our previous workon WT Pin1 functional dynamics in the presenceand absence of inhibitors and substrates has led us to propose anadditional mechanism for WW domain influence on PPIase activity. Specifically,we proposed that the WW domain also acts as an allosteric effectormolecule of the PPIase domain. The allosteric binding site involvesthe interdomain contact region identified in this study. The mechanismthat communicates changes at the interdomain contact region to thedistal PPIase catalytic site involves propagated changes in flexibilityamong the intervening residues. These flexibility changes manifestas a loss of subnanosecond side chain flexibility along a conduitof conserved hydrophobic residues linking the interdomain interfaceto the active site.^1,2

If this dynamic allostericexplanation is tenable, then weakening the interdomain contact viathe I28A mutation should produce a different response in side-chaindynamics upon Cdc25C substrate binding. This is what we observe (Figure 7, middle and bottom panels). Critically, I28A lackskey features of the WT dynamic conduit, such as the loss of side-chainflexibility for the PPIase active site residues L60 and L61. In fact,L60 becomes more flexible (Figure 7, middlepanel). The colored structure (PDB id 1PIN) at the bottom of Figure 7 further underscores these differences. This differentialdynamic response is reinforced by the corresponding ¹⁵Nbackbone dynamics; specifically, I28A showed an enhancement of backbonesubnanosecond flexibility for S58, V62, and C113 upon Cdc25C substratebinding (Figure 6) that is absent in the WT.The side chain and backbone results above suggest that the interdomaininterface is a critical set of interactions that enable dynamic allostericregulation of the PPIase domain by the WW domain.

Long-RangeInteractions within the WW Domain

I28A weakenedboth interdomain contact and WW domain binding affinity to the Cdc25Cphosphopeptide. This joint effect is intriguing because I28 does notdirectly contact substrate; rather, it is on the opposite side ofthe WW domain loop mediating substrate binding, Loop I (S16–R21).The fact that the binding affinity at Loop I is sensitive to a mutationat the far end of the domain points to a yet unremarked mechanismfor long-range Loop I–Loop II communication within the Pin1WW domain.

We speculate that this long-range communication derivesfrom a network of short-range inter-residue interactions within theWW domain. This speculation derives from our unexpected observationthat I28A causes changes in mobility beyond its host Loop II and extendsto K13 and R14 in the β1′-strand (Figure 5). These long-range perturbations become comprehensible whenthe Loop II hydrogen bond network in the 1PIN crystal structures is examined (Figure 8).^22,53 Of particular interest are hydrogenbonds from the I28A backbone NH to side chains of N26. N26 is highlyconserved across Pin1 homologues¹⁷ andmakes multiple hydrogen bonds that stabilize Loop II and link it toβ1′ (cf. Figure 8). In I28A, N26shows large backbone chemical shift perturbations (Figure 4, Figures S1 of the SupportingInformation) and enhanced exchange dynamics (amplified J_eff^NH(0) in Figure 6) that are absent in WT. Thus,although we did not mutate N26 directly, we have nevertheless changedits local mobility by mutating one of its hydrogen bond partners,I28. Conceivably, the shorter Ala side chain in I28A could decreaseside chain steric contacts, both with the PPIase domain and withinLoop II itself. Indeed, the perturbations in ¹³Cα/βchemical shifts for Loop II (apo I28A versus apo WT) suggest localstructural perturbations to Loop II, consistent with this notion (FigureS1 of the Supporting Information). Thiscould enable greater backbone mobility at position 28, which wouldthen propagate to N26 and more remote sites, such as K13 and R14,via the network of backbone and side-chain hydrogen bonds. Thus, thelong-range mobility perturbations stimulated by I28A make it reasonableto contemplate a network of short-range interactions within the WWdomain that could couple perturbations at Loop II to Loop I.

Figure 8

Loop II hydrogenbond pairings of Pin1 WW domain within full-lengthWT Pin1 (PDB id 1PIN). Solid line black arrows represent backbone–backbone hydrogenbonds, whereas dotted black arrows represent backbone–side-chainhydrogen bonds. The backbone NH of I28 makes hydrogen bonds to theside chain of N26.

If this intra-WW domainnetwork is corroborated by subsequent experiments,it would mean that substrate binding to the WW domain, and its allostericinfluence on the PPIase domain, are themselves coupled phenomena.It would also justify the hypothesis of a larger network of interactingresidues that couple binding events at the WW domain Loop I (S16–R21)all the way to the PPIase active site, with the interdomain contactsurface as a crucial intermediary.

Coevolving Residues

By itself, I28 is not a highlyconserved residue across Pin1 homologues.¹⁷ Nevertheless, our results show that I28 participates in inter-residueinteractions that sustain the weak contacts between the WW and PPIasedomain (e.g., the large chemical shift perturbations at PPIase domainresidues S138–R142 in Figure 3). Thus,we might expect that I28 would emerge in bioinformatics analyses aimedat finding pairs of coevolving residues. An example is the “proteinsector” analysis of Ranganathan and co-workers, which identifiessectors of coevolving residues based on their statistical couplinganalysis (SCA) of multiple sequence alignments (MSA).⁵⁴ Our initial application of this sector/SCA to Pin1 revealsI28/A140 as a coevolving pair. This pair would be consistent withthe interdomain contacts identified above (e.g., chemical shift perturbationsin Figure 3, side-chain dynamic changes Figure 7), and form the basis for future double mutant studies.

Interdomain Contact Supports Interdomain Allostery in Pin1

In summary, our I28A results reveal that the WW domain Loop II(H27–N30) is critical for establishing transient interdomaincontacts with the PPIase domain. Moreover, these contacts influencethe distribution of conformations sampled by the PPIase domain. Evidencefor this influence consists of the perturbations to backbone chemicalshifts, backbone/side-chain mobility, substrate binding affinity,and isomerase activity documented above. These results show that interdomaincontact alters the internal properties of the PPIase domain and thusstrengthen our hypothesis for allosteric communication between theinterdomain interface and the distal active site.

We shouldreiterate that although this investigation reveals Pin1 interdomainallostery mainly through changes in protein dynamics parameters, itdoes not exclude the possibility of joint changes in local conformation.Indeed, the backbone chemical shift perturbations indicate that, althoughthe I28A mutation preserves the overall WT fold, it may also instigatelocal structural changes within the PPIase domain. This possibilityis consistent with our main point: a WW domain mutation (I28A) atthe domain interface can perturb the conformational sampling of thePPIase domain in such a way that it alters local flexibility, structure,or both. All three scenarios would be consequences of interdomainallostery.

By combining our I28A results with our previous ones,we envisionthe following underlying scenario. The Pin1 interdomain interactions,although weak, influence the ensemble of conformations sampled byboth domains. From the perspective of the PPIase domain, the WW domainacts not only as a binding module but also as an allosteric effectormolecule. WW domain contact with the PPIase domain perturbs the conformationalsampling by the PPIase domain. These changes in conformational sampling,although stimulated at the domain interface, can propagate away fromthat interface to the remote catalytic site via correlated internalmotions within the PPIase domain. These correlated motions include(but are not limited to) the side chain motions whose perturbationsmanifest as the dynamic conduit. The results are not gross structuralchanges but rather subtle changes in local conformation and flexibilityat the PPIase catalytic site that fine-tune binding affinity and isomerizationactivity. Substrate binding stabilizes the subset of conformationsinvolving more intimate interdomain contact and thereby tunes bindingaffinity and activity. The exact manner of tuning doubtless dependson the details of substrate composition (e.g., residues flanking thepS/T–P segments).

To further investigate the above scenario,we require further mutationstudies. In particular, we need mutations that perturb interdomaincontact, exclusively, without perturbing the binding affinity of WWdomain residues. These mutations could involve those of the PPIaseside of the domain interface, and modifications of the flexible linker.Such work is in progress.

Modular proteins are replete in biochemicalnetworks maintainingthe cell cycle.⁴ The weak interdomain interactionsinvestigated here for Pin1 may be present in other modular systemsand, perhaps, play similar functional roles. Perturbing these interactionssystematically, via small molecule ligands, may be a promising approachfor advancing our understanding of the molecules regulating cell growth.Also, as mentioned in the introduction, functional interdomain interactionsare attractive target sites for the design of allosteric inhibitors.^6,7 Conceivably, fragment-based approaches that target both the catalyticsite and interdomain interfaces may enhance target specificity.