Dramatic Structural Changes Resulting from the Loss of a Crucial Hydrogen Bond in the Hinge Region Involved in C-Terminal Helix Swapping in SurE: A Survival Protein from Salmonella typhimurium

Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two β strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 Å and 2.35 Å resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2– H234 NE2 hydrogen bond (2.89 Å in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE.     

Circular permutation and deletion studies of myoglobin indicate that the correct position of its N-terminus is required for native stability and solubility but not for native-like heme binding and folding

We studied the effect of deleted and circularly permuted mutations in sperm whale myoglobin and present here results on three classes of mutants: (i) a deletion mutant, Mb(1)(-)(99), in which the C-terminal helices, G and H, were removed; (ii) two circular permutations, Mb-B_GHA, in which helix B is N-terminal and helix A is C-terminal, and Mb-C_GHAB, in which helix C is N-terminal and helices A and B are C-terminal; and (iii) a deleted circular permutation, Mb-HAB_F, in which helix H is N-terminal, helix F is C-terminal, and helix G is deleted. The conformational characteristics of the apo and holo forms of these mutants were determined at neutral pH, by spectroscopic and hydrodynamic methods. The apo form of the deleted and permuted mutants exhibited a stronger tendency to aggregate and had lower ellipticity than the wild type. The mutants retained the ability to bind heme, but only the circularly permuted holoproteins had native-like heme binding and folding. These results agree with the theory that myoglobin has a central core that is able to bind heme, but also indicate that the presence of N- and C-terminal helices is necessary for native-like heme pocket formation. Because the holopermuteins were less stable than the wild-type protein and aggregated, we propose that the native position of the N-terminus is important for the precise structural architecture of myoglobin.     

Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.     

Dynamic properties of the N-terminal swapped dimer of ribonuclease A

Bovine pancreatic ribonuclease (RNase A) forms two 3-dimensional domain-swapped dimers with different quaternary structures. One dimer is characterized by the swapping of the C-terminal region (C-Dimer) and presents a rather loose structure. The other dimer (N-Dimer) exhibits a very compact structure with exchange of the N-terminal helix. Here we report the results of a molecular dynamics/essential dynamics (MD/ED) study carried out on the N-Dimer. This investigation, which represents the first MD/ED analysis on a three-dimensional domain-swapped enzyme, provides information on the dynamic properties of the active site residues as well as on the global motions of the dimer subunits. In particular, the analysis of the flexibility of the active site residues agrees well with recent crystallographic and site-directed mutagenesis studies on monomeric RNase A, thus indicating that domain swapping does not affect the dynamics of the active sites. A slight but significant rearrangement of N-Dimer quaternary structure, favored by the formation of additional hydrogen bonds at subunit interface, has been observed during the MD simulation. The analysis of collective movements reveals that each subunit of the dimer retains the functional breathing motion observed for RNase A. Interestingly, the breathing motion of the two subunits is dynamically coupled, as they open and close in phase. These correlated motions indicate the presence of active site intercommunications in this dimer. On these bases, we propose a speculative mechanism that may explain negative cooperativity in systems preserving structural symmetry during the allosteric transitions.     

Conformational properties of the disease-causing Z variant of α1-antitrypsin revealed by theory and experiment

The human serine protease inhibitor (serpin) α-1 antitrypsin (α1-AT) protects tissues from proteases of inflammatory cells. The most common disease-causing mutation in α1-AT is the Z-mutation (E342K) that results in an increased propensity of α1-AT to polymerize in the ER of hepatocytes, leading to a lack of secretion into the circulation. The structural consequences of this mutation, however, remain elusive. We report a comparative molecular dynamics investigation of the native states of wild-type and Z α1-AT, revealing a striking contrast between their structures and dynamics in the breach region at the top of β-sheet A, which is closed in the wild-type simulations but open in the Z form. Our findings are consistent with experimental observations, notably the increased solvent exposure of buried residues in the breach region in Z, as well as polymerization via domain swapping, whereby the reactive center loop is rapidly inserted into an open A-sheet before proper folding of the C-terminal β-strands, allowing C-terminal domain swapping with a neighboring molecule. Taken together, our experimental and simulation data imply that mutations at residue 342 that either stabilize an open form of the top of β-sheet A or increase the local flexibility in this region, may favor polymerization and hence aggregation.     

Molecular dynamics modeling of tubulin C-terminal tail interactions with the microtubule surface

Tubulin, an α/β heterodimer, has had most of its 3D structure analyzed; however, the carboxy (C)-termini remain elusive. Importantly, the C-termini play critical roles in regulating microtubule structure and function. They are sites of most of the post-translational modifications of tubulin and interaction sites with molecular motors and microtubule-associated proteins. Simulated annealing was used in our molecular dynamics modeling to predict the interactions of the C-terminal tails with the tubulin dimer. We examined differences in their flexibility, interactions with the body of tubulin, and the existence of structural motifs. We found that the α-tubulin tail interacts with the H11 helix of β-tubulin, and the β-tubulin tail interacts with the H11 helix of α-tubulin. Tail domains and H10/B9 loops interact with each other and compete for interactions with positively-charged residues of the H11 helix on the neighboring monomer. In a simulation in which α-tubulin's H10/B9 loop switches on sub-nanosecond intervals between interactions with the C-terminal tail of α-tubulin and the H11 helix of β-tubulin, the intermediate domain of α-tubulin showed more fluctuations compared to those in the other simulations, indicating that tail domains may cause shifts in the position of this domain. This suggests that C-termini may affect the conformation of the tubulin dimer which may explain their essential function in microtubule formation and effects on ligand binding to microtubules. Our modeling also provides evidence for a disordered-helical/helical double-state system of the T3/H3 region of the microtubule, which could be linked to depolymerization following GTP hydrolysis.     

SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer

SxIP is a microtubule tip localizing signal found in many +TIP proteins that bind to the hydrophobic cavity of the C-terminal domain of end binding protein 1 (EB1) and then positively regulate the microtubule plus-end tracking of EBs. However, the exact mechanism of microtubule activation of EBs in the presence of SxIP signaling motif is not known. Here, we studied the effect of SxIP peptide on the native conformation of EB1 in solution. Using various NMR experiments, we found that SxIP peptide promoted the dissociation of natively formed EB1 dimer. We also discovered that I224A mutation of EB1 resulted in an unfolded C-terminal domain, which upon binding with the SxIP motif folded to its native structure. Molecular dynamics simulations also confirmed the relative structural stability of EB1 monomer in the SxIP bound state. Residual dipolar couplings and heteronuclear NOE analysis suggested that the binding of SxIP peptide at the C-terminal domain of EB1 decreased the dynamics and conformational flexibility of the N-terminal domain involved in EB1-microtubule interaction. The SxIP-induced disruption of the dimeric interactions in EB1, coupled with the reduction in conformational flexibility of the N-terminal domain of EB1, might facilitate the microtubule association of EB1.     

Proton magnetic resonance characterization of the dynamic stability of the heme pocket in myoglobin by the exchange behavior of the labile proton of the proximal histidyl imidazole.

The assigned exchangeable proton signals in the proton nuclear magnetic resonance spectra of sperm whale deoxy and Met-cyano myoglobin in H2O solution were found to exhibit pH-dependent saturation transfer from the bulk water, which allowed determination of the kinetics and mechanism of the labile proton exchange with solvent. The exchange rates are base catalyzed for both protein forms, with the rate eight times faster in Met-cyano than in deoxy myoglobin. The exchange rate is taken as a measure of the magnitude of the fluctuation in the protein conformation near the heme cavity. On the basis of tritium exchange methods, the greater stability of the unligated relative to the ligated state in myoglobin has also been reported for hemoglobin. The present study, however, localizes the differential kinetic stability on the F helix whose flexibility has been implicated in the mechanism of cooperativity. The observation that filling the hydrophobic vacancy on the proximal side of the heme near the proximal histidine in Met-cyano myoglobin wih cyclopropane increases the proton lability argues against the role for this hole in facilitating the flexibility of the F helix in the native protein.     

Structures of the two 3D domain-swapped RNase A trimers

When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.     

The Distinct Conformational Dynamics of K-Ras and H-Ras A59G

Ras proteins regulate signaling cascades crucial for cell proliferation and differentiation by switching between GTP- and GDP-bound conformations. Distinct Ras isoforms have unique physiological functions with individual isoforms associated with different cancers and developmental diseases. Given the small structural differences among isoforms and mutants, it is currently unclear how these functional differences and aberrant properties arise. Here we investigate whether the subtle differences among isoforms and mutants are associated with detectable dynamical differences. Extensive molecular dynamics simulations reveal that wild-type K-Ras and mutant H-Ras A59G are intrinsically more dynamic than wild-type H-Ras. The crucial switch 1 and switch 2 regions along with loop 3, helix 3, and loop 7 contribute to this enhanced flexibility. Removing the gamma-phosphate of the bound GTP from the structure of A59G led to a spontaneous GTP-to-GDP conformational transition in a 20-ns unbiased simulation. The switch 1 and 2 regions exhibit enhanced flexibility and correlated motion when compared to non-transitioning wild-type H-Ras over a similar timeframe. Correlated motions between loop 3 and helix 5 of wild-type H-Ras are absent in the mutant A59G reflecting the enhanced dynamics of the loop 3 region. Taken together with earlier findings, these results suggest the existence of a lower energetic barrier between GTP and GDP states of the mutant. Molecular dynamics simulations combined with principal component analysis of available Ras crystallographic structures can be used to discriminate ligand- and sequence-based dynamic perturbations with potential functional implications. Furthermore, the identification of specific conformations associated with distinct Ras isoforms and mutants provides useful information for efforts that attempt to selectively interfere with the aberrant functions of these species.     

Molecular dynamics simulations of transducin: interdomain and front to back communication in activation and nucleotide exchange

The dynamic events that underlie the nucleotide exchange process for the Galpha subunit of transducin (Galpha(t)) were studied with nanosecond time-scale molecular dynamics simulations. The modeled systems include the active and inactive forms of the wild-type Galpha(t) and three of its mutants (GDP-bound form only): F332A, A322S, and Q326A that are known to exhibit various degrees of enhancement of their basal and receptor-catalyzed rates of nucleotide exchange (150-fold, 70-fold and WT-like, respectively). The results of these computational experiments reveal a number of nucleotide-dependent structural and dynamic changes (involving the alpha(B)-alpha(C) loop, the inter-domain orientation of the helical and GTPase domains and the alpha(5) helix) that were not observed in the various crystal structures of Galpha(t). Notably, the results show the existence of a front to back communication device (involving the beta(2)-beta(3) hairpin, the alpha(1) helix and the alpha(5) helix), strategically located near all elements susceptible to be involved in receptor-mediated activation/nucleotide exchange. The wild-type simulations suggest that the dynamic interplay between the elements of this device would be critical for the activation of the Galpha(t) subunit. This inference is confirmed by the results of the computational experiments on the mutants that show that even in their GDP-bound forms, the A322S and F332A mutants acquire an "active-like" structure and dynamics phenotype. The same is not true for the Q326A mutant whose structural and dynamic properties remain similar to those of the GDP-bound WT. Taken together the results suggest a nucleotide exchange mechanism, analogous to that found in the Arf family GTPases, in which a partially activated state, achievable from a receptor-mediated action of the front to back communication device either by displacement of the C-terminal alpha(5) helix, of the N-terminal alpha(N) helix, or of the Gbetagamma subunit, could precede the dissociation of GDP from the native Galpha subunit.     

Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: structural evidence for cysteine binding and lack of interactions with serine acetyl transferase

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.     

The dynamics of camphor in the cytochrome P450 CYP101D2

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate‐free) and camphor‐soaked forms have open conformations. Furthermore, two other potential camphor‐binding sites were also identified from electron densities in the camphor‐soaked structure, one being located in the access channel and the other in a cavity on the surface near the F‐helix side of the F‐G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor‐bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor‐bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit.     

Investigation of Domain Motions in Bacteriophage T4 Lysozyme

Abstract

Hinge-bending in T4 lysozyme has been inferred from single amino acid mutant crystalline allomorphs by Matthews and coworkers. This raises an important question: are the different conformers in the unit cell artifacts of crystal packing forces, or do they represent different solution state structures? The objective of this theoretical study is to determine whether domain motions and hinge-bending could be simulated in T4 lysozyme using molecular dynamics. An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented. Molecular dynamics calculations were computed using the Discover software package (Biosym Technologies). All hydrogen atoms were modeled explicitly with the inclusion of all 152 crystallographic waters at a temperature of 300 K. The native T4 lysozyme molecular dynamics simulation demonstrated hinge-bending in the protein. Relative domain motions between the N-terminal and C-terminal domains were evident. The enzyme hinge bending sites resulted from small changes in backbone atom conformations over several residues rather than rotation about a single bound. Two hinge loci were found in the simulation. One locus comprises residues 8–14 near the C-terminal of the A helix; the other site, residues 77–83 near the C-terminal of the C helix. Comparison of several snapshot structures from the dynamics trajectory clearly illustrates domain motions between the two lysozyme lobes. Time correlated atomic motions in the protein were analyzed using a dynamical cross-correlation map. We found a high degree of correlated atomic motions in each of the domains and, to a lesser extent, anticorrelated motions between the two domains. We also found that the hairpin loop in the N-terminal lobe (residues 19–24) acted as a mobile ‘flap’ and exhibited highly correlated dynamic motions across the cleft of the active site, especially with residue 142.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

Energetic Frustration of Apomyoglobin Folding: Role of the B Helix

Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the GH turn region in the G23A/G25A mutant, suggesting that stabilization of the B helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild-type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.     

Intrinsic flexibility of NLRP pyrin domains is a key factor in their conformational dynamics,fold stability,and dimerization

Nucleotide‐binding domain leucine‐rich repeat‐containing receptors (NLRs) are key proteins in the innate immune system. The 14 members of the NLRP family of NLRs contain an N‐terminal pyrin domain which is central for complex formation and signal transduction. Recently, X‐ray structures of NLRP14 revealed an unexpected rearrangement of the α5/6 stem‐helix of the pyrin domain allowing a novel symmetric dimerization mode. We characterize the conformational transitions underlying NLRP oligomerization using molecular dynamics simulations. We describe conformational stability of native NLRP14 and mutants in their monomeric and dimeric states and compare them to NLRP4, a representative of a native pyrin domain fold. Thereby, we characterize the interplay of conformational dynamics, fold stability, and dimerization in NLRP pyrin domains. We show that intrinsic flexibility of NLRP pyrin domains is a key factor influencing their behavior in physiological conditions. Additionally, we provide further evidence for the crucial importance of a charge relay system within NLRPs that critically influences their conformational ensemble in solution.     

Propensity for C-terminal domain swapping correlates with increased regional flexibility in the C-terminus of RNase A

Domain swapping is a type of oligomerization in which monomeric proteins exchange a structural element, resulting in oligomers whose subunits recapitulate the native, monomeric fold. It has been implicated as a potential mechanism for protein aggregation, which provides a strong impetus to understand the structural determinants and folding mechanisms that trigger domain swapping. Bovine pancreatic ribonuclease A (RNase A) is a well-studied protein known to domain swap under extreme conditions, such as lyophilization from acetic acid. The major domain-swapped dimer form of RNase A exchanges a β-strand at its C-terminus to form a C-terminal domain-swapped dimer. To study the mechanism by which C-terminal swapping occurs, we used a variant of RNase A containing a P114G mutation that readily domain swaps under physiological conditions. Using NMR and hydrogen-deuterium exchange, we find that the P114G variant has decreased protection from hydrogen exchange compared to the wild-type protein near the C-terminal hinge region. Our results suggest that domain swapping occurs via a local high-energy fluctuation at the C-terminus.     

Structure and function of the C-terminal domain of methionyl-tRNA synthetase

The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain. The minimal MetRS behaves as a monomer. In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide. Upon truncation of this C-terminal domain, subunits dissociate irreversibly. Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied. It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea. The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS. Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity. However, dimer formation is required to evidence the gain in affinity.