[show abstract][hide abstract] ABSTRACT: Rev1 is a Y-family translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap filling. In the process of TLS high-fidelity replicative DNA polymerases stalled by DNA damage are replaced by error-prone TLS enzymes responsible for the majority of mutagenesis in eukaryotic cells. The polymerase exchange that gains low-fidelity TLS polymerases access to DNA is mediated by their interactions with proliferating cell nuclear antigen (PCNA). Rev1 stands alone from other Y-family TLS enzymes since it lacks the consensus PCNA-interacting protein box (PIP-box) motif, instead utilizing other modular domains for PCNA binding. Here we report solution NMR structure of an 11 kDa BRCA1 C-terminus (BRCT) domain from S. cerevisiae Rev1, and demonstrate with the use of TROSY NMR methods that Rev1-BRCT domain directly interacts with an 87 kDa PCNA in solution. The domain adopts α/β fold (β1-α1-β2-β3-α2-β4-α3-α4) typical for BRCT domain superfamily. PCNA-binding interface of the Rev1-BRCT domain comprises conserved residues of the outer surface of the α1 helix, α1-β1, β2-β3 and β3-α2 loops. On the other hand, Rev1-BRCT binds to the inter-domain region of PCNA that overlaps with the binding site for the PIP-box motif. Furthermore, Rev1-BRCT domain bound to PCNA can be displaced by increasing amounts of the PIP-box peptide from TLS DNA polymerase polη, suggesting that Rev1-BRCT and polη PIP-box interactions with the same PCNA monomer are mutually exclusive. These results provide structural insights into PCNA recognition by TLS DNA polymerases that help better understand TLS regulation in eukaryotes.
Journal of Molecular Biology 06/2013; · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (polς). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the 'inserter' polη and Rev7 subunit of the 'extender' polς, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS. STRUCTURED SUMMARY OF PROTEIN INTERACTIONS: REV1, REV3 and REV7physically interact by nuclear magnetic resonance (View interaction), molecular sieving (View interaction) and isothermal titration calorimetry (View interaction). REV3 and REV7bind by molecular sieving (View interaction) REV1 and Polη-RIR peptidebind by nuclear magnetic resonance (View interaction) REV1, REV3, REV7 and Polη-RIR peptidephysically interact by nuclear magnetic resonance (View interaction).
[show abstract][hide abstract] ABSTRACT: Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.
[show abstract][hide abstract] ABSTRACT: Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module-the FF domain-with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1·10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.
Proceedings of the National Academy of Sciences 05/2012; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have recently reported the atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 [Korzhnev, D. M.; Religa, T. L.; Banachewicz, W.; Fersht, A. R.; Kay, L.E. Science 2011, 329, 1312-1316]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints of the invisible intermediate state measured using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the database structure determination program, CS-Rosetta. As a cross-validation of the structure so produced, we present here the solution structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by mutagenesis that destabilizes the native state. The relaxation dispersion/CS-Rosetta structures of the intermediate are within 2 Å of those of the mimic, with the nonnative interactions in the intermediate also observed in the mimic. This strongly confirms the structure of the FF domain folding intermediate, in particular, and validates the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.
The Journal of Physical Chemistry B 12/2011; 116(23):6637-44. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several all-helical single-domain proteins have been shown to fold rapidly (microsecond time scale) to a compact intermediate state and subsequently rearrange more slowly to the native conformation. An understanding of this process has been hindered by difficulties in experimental studies of intermediates in cases where they are both low-populated and only transiently formed. One such example is provided by the on-pathway folding intermediate of the small four-helix bundle FF domain from HYPA/FBP11 that is populated at several percent with a millisecond lifetime at room temperature. Here we have studied the L24A mutant that has been shown previously to form nonnative interactions in the folding transition state. A suite of Carr-Purcell-Meiboom-Gill relaxation dispersion NMR experiments have been used to measure backbone chemical shifts and amide bond vector orientations of the invisible folding intermediate that form the input restraints in calculations of atomic resolution models of its structure. Despite the fact that the intermediate structure has many features that are similar to that of the native state, a set of nonnative contacts is observed that is even more extensive than noted previously for the wild-type (WT) folding intermediate. Such nonnative interactions, which must be broken prior to adoption of the native conformation, explain why the transition from the intermediate state to the native conformer (millisecond time scale) is significantly slower than from the unfolded ensemble to the intermediate and why the L24A mutant folds more slowly than the WT.
Journal of the American Chemical Society 06/2011; 133(28):10974-82. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Wilson disease protein (ATP7B) is a copper-transporting member of the P-type ATPase superfamily, which plays a central role in copper homeostasis and interacts with the copper chaperone Atox1. The N-terminus of ATP7B is comprised of six copper-binding domains (WCBDs), each capable of binding one copper atom in the +1 oxidation state. To better understand the regulatory effect of copper binding to these domains, we have performed NMR characterization of WCBD4-6 (domains 4-6 of ATP7B). (15)N relaxation measurements on the apo and Cu(I)-bound WCBD4-6 show that there is no dramatic change in the dynamic properties of this three-domain construct; the linker between domains 4 and 5 remains flexible, domains 5 and 6 do not form a completely rigid dimer but rather have some flexibility with respect to each other, and there is minimal change in the relative orientation of the domains in the two states. We also show that, contrary to previous reports, the protein-protein interaction between Atox1 and the copper-binding domains takes place even in the absence of copper. Comparison of apo and Cu(I)-bound spectra of WCBD1-6 shows that binding of Cu(I) does not induce the formation of a unit that tumbles as a single entity, consistent with our results for WCBD4-6. We propose that copper transfer to and between the N-terminal domains of the Wilson Cu-ATPase occurs via protein interactions that are facilitated by the flexibility of the linkers and the motional freedom of the domains with respect to each other.
[show abstract][hide abstract] ABSTRACT: Proteins can sample conformational states that are critical for function but are seldom detected directly because of their low occupancies and short lifetimes. In this work, we used chemical shifts and bond-vector orientation constraints obtained from nuclear magnetic resonance relaxation dispersion spectroscopy, in concert with a chemical shift-based method for structure elucidation, to determine an atomic-resolution structure of an "invisible" folding intermediate of a small protein module: the FF domain. The structure reveals non-native elements preventing formation of the native conformation in the carboxyl-terminal part of the protein. This is consistent with the kinetics of folding in which a well-structured intermediate forms rapidly and then rearranges slowly to the native state. The approach introduces a general strategy for structure determination of low-populated and transiently formed protein states.
[show abstract][hide abstract] ABSTRACT: NMR relaxation dispersion spectroscopy is a powerful method for studying protein conformational dynamics whereby visible, ground and invisible, excited conformers interconvert on the millisecond time-scale. In addition to providing kinetics and thermodynamics parameters of the exchange process, the CPMG dispersion experiment also allows extraction of the absolute values of the chemical shift differences between interconverting states, /Delta(omega)/, opening the way for structure determination of excited state conformers. Central to the goal of structural analysis is the availability of the chemical shifts of the excited state that can only be obtained once the signs of Delta(omega) are known. Herein we describe a very simple method for determining the signs of (1)H(N) Delta(omega) values based on a comparison of peak positions in the directly detected dimensions of a pair of (1)H(N)-(15)N correlation maps recorded at different static magnetic fields. The utility of the approach is demonstrated for three proteins that undergo millisecond time-scale conformational rearrangements. Although the method provides fewer signs than previously published techniques it does have a number of strengths: (1) Data sets needed for analysis are typically available from other experiments, such as those required for measuring signs of (15)N Delta(omega) values, thus requiring no additional experimental time, (2) acquisition times in the critical detection dimension can be as long as necessary and (3) the signs obtained can be used to cross-validate those from other approaches.
Journal of Biomolecular NMR 06/2010; 47(2):135-41. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible 'excited' conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Deltavarpi|), that inform on the structural properties of the excited state(s). The sign of Deltavarpi is, however, not available from CPMG data. Here we present one-dimensional NMR experiments for measuring the signs of (1)H(N) and (13)C(alpha) Deltavarpi values using weak off-resonance R (1rho ) relaxation measurements, extending the spin-lock approach beyond previous applications focusing on the signs of (15)N and (1)H(alpha) shift differences. The accuracy of the method is established by using an exchanging system where the invisible, excited state can be converted to the visible, ground state by altering conditions so that the signs of Deltavarpi values obtained from the spin-lock approach can be validated with those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring the signs of chemical shift differences. For the Abp1p and Fyn SH3 domains considered here it is found that while H(S/M)QC measurements provide signs for more residues than the spin-lock data, the two different methodologies are complementary, so that combining both approaches frequently produces signs for more residues than when the H(S/M)QC method is used alone.
Journal of Biomolecular NMR 03/2010; 46(3):205-16. · 2.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Marginally and transiently populated conformational states of biomolecules can play important functional roles in biochemical processes. It is of significant interest, therefore, to develop tools for characterizing the structural and dynamical properties of these excited states. One recent development has been the emergence of spin-state-selective relaxation dispersion methods for quantifying dipolar vector orientations in invisible excited-state conformers through measurement of residual dipolar couplings (RDCs). Particularly powerful are 1HN-(15)N RDCs that can be measured with high sensitivity on fractionally aligned, deuterated, uniformly 15N-labeled protein samples. Fractional alignment also produces nonzero 1HN-(1)HN dipolar couplings. These can be problematic for the extraction of robust 1HN-(15)N RDC values, and hence amide bond vector orientations, in cases where the amide proton of interest and a proximal amide proton have small chemical shift differences and a significant 1HN-(1)HN dipolar coupling. Here, we show that while this strong coupling effect leads to aberrant relaxation dispersion profiles, extracted excited-state 1HN-(15)N RDCs are for the most part only marginally affected. Experimental examples of such aberrant profiles are provided, as well as a theoretical consideration of the influence of this strong coupling effect and numerical simulations that assess its impact on extracted parameters.
The Journal of Physical Chemistry B 08/2009; 113(29):9968-77. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Surfaces of many binding domains are plastic, enabling them to interact with multiple targets. An understanding of how they bind and recognize their partners is therefore predicated on characterizing such dynamic interfaces. Yet, these interfaces are difficult to study by standard biophysical techniques that often 'freeze' out conformations or that produce data averaged over an ensemble of conformers. In this study, we used NMR spectroscopy to study the interaction between the C-terminal SH3 domain of CIN85 and ubiquitin that involves the 'classical' binding sites of these proteins. Notably, chemical shift titration data of one target with another and relaxation dispersion data that report on millisecond time scale exchange processes are both well fit to a simple binding model in which free protein is in equilibrium with a single bound conformation. However, dissociation constants and chemical shift differences between free and bound states measured from both classes of experiment are in disagreement. It is shown that the data can be reconciled by considering three-state binding models involving two distinct bound conformations. By combining titration and dispersion data, kinetic and thermodynamic parameters of the three-state binding reaction are obtained along with chemical shifts for each state. A picture emerges in which one bound conformer has increased entropy and enthalpy relative to the second and chemical shifts similar to that of the free state, suggesting a less packed interface. This study provides an example of the interplay between entropy and enthalpy to fine-tune molecular interactions involving the same binding surfaces.
Journal of Molecular Biology 01/2009; 386(2):391-405. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biological function depends on molecular dynamics that lead to excursions from highly populated ground states to much less populated excited states. The low populations and the transient formation of such excited states render them invisible to the conventional methods of structural biology. Thus, while detailed pictures of ground-state structures of biomolecules have emerged over the years, largely through X-ray diffraction and solution nuclear magnetic resonance (NMR) spectroscopy studies, much less structural data has been accumulated on the conformational properties of the invisible excited states that are necessary to fully explain function. NMR spectroscopy is a powerful tool for studying conformational dynamics because it is sensitive to dynamics over a wide range of time scales, extending from picoseconds to seconds and because information is, in principle, available at nearly every position in the molecule. Here an NMR method for quantifying millisecond time scale dynamics that involve transitions between different molecular conformations is described. The basic experimental approach, termed relaxation dispersion NMR spectroscopy, is outlined to provide the reader with an intuitive feel for the technology. A variety of different experiments that probe conformational exchange at different sites in proteins are described, including a brief summary of data-fitting procedures to extract both the kinetic and thermodynamic properties of the exchange process and the structural features of the invisible excited states along the exchange pathway. It is shown that the methodology facilitates detection of intermediates and other excited states that are populated at low levels, 0.5% or higher, that cannot be observed directly in spectra, so long as they exchange with the observable ground state of the protein on the millisecond time scale. The power of the methodology is illustrated by a detailed application to the study of protein folding of the small modular SH3 domain. The kinetics and thermodynamics that describe the folding of this domain have been characterized through the effects of temperature, pressure, side-chain deuteration, and mutation, and the structural features of a low-populated folding intermediate have been assessed. Despite the fact that many previous studies have shown that SH3 domains fold via a two-state mechanism, the NMR methods presented unequivocally establish the presence of an on-pathway folding intermediate. The unique capabilities of NMR relaxation dispersion follow from the fact that large numbers of residues can be probed individually in a single experiment. By contrast, many other forms of spectroscopy monitor properties that are averaged over all residues in the molecule or that make use of only one or two reporters. The NMR methodology is not limited to protein folding, and applications to enzymatic catalysis, binding, and molecular recognition are beginning to emerge.
Accounts of Chemical Research 04/2008; 41(3):442-51. · 20.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.
Proceedings of the National Academy of Sciences 10/2007; 104(36):14336-41. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.
Journal of Molecular Biology 09/2007; 372(2):497-512. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: NMR spectroscopy and computer simulations were used to examine changes in chemical shifts and in dynamics of the ribonuclease barnase that result upon binding to its natural inhibitor barstar. Although the spatial structures of free and bound barnase are very similar, binding results in changes of the dynamics of both fast side-chains, as revealed by (2)H relaxation measurements, and NMR chemical shifts in an extended beta-sheet that is located far from the binding interface. Both side-chain dynamics and chemical shifts are sensitive to variations in the ensemble populations of the inter-converting molecular states, which can escape direct structural observation. Molecular dynamics simulations of free barnase and barnase in complex with barstar, as well as a normal mode analysis of barnase using a Gaussian network model, reveal relatively rigid domains that are separated by the extended beta-sheet mentioned above. The observed changes in NMR parameters upon ligation can thus be rationalized in terms of changes in inter-domain dynamics and in populations of exchanging states, without measurable structural changes. This provides an alternative model for the propagation of a molecular response to ligand binding across a protein that is based exclusively on changes in dynamics.
Journal of Molecular Biology 05/2007; 367(4):1079-92. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Src homology 3 (SH3) domains are small modules that are thought to fold via a two-state mechanism, without the accumulation of significant populations of intermediate states. Relaxation dispersion NMR studies of the folding of G48V and G48M mutants of the Fyn SH3 domain have established that, at least for these modules, folding proceeds through the formation of a transient on-pathway intermediate with an equilibrium population of 1-2% that can be readily detected [Korzhnev, D. M., et al. (2004) Nature 430, 586-590]. To investigate the generality of this result, we present an (15)N relaxation dispersion NMR study of a pair of additional SH3 domains, including a G48V mutant of a stabilized Abp1p SH3 domain that shares 36% sequence identity with the Fyn SH3 module, and a A39V/N53P/V55L mutant Fyn SH3 domain. A transient folding intermediate is detected for both of the proteins studied here, and the dispersion data are well fit to a folding model of the form F <--> I <--> U, where F, I, and U correspond to folded, intermediate, and unfolded states, respectively. The temperature dependencies of the folding/unfolding rate constants were obtained so that the thermodynamic properties of each of F, I, and U could be established. The detection of I states in folding pathways of all SH3 domains examined to date via relaxation dispersion NMR spectroscopy indicates that such intermediates may well be a conserved feature in the folding of such domains in general but that their transient nature along with their low population makes detection difficult using more well-established approaches to the study of folding.