Alaji Bah

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein’s structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the ‘invisible’, excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein’s function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch.

Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function. In principle, the function of intrinsically disordered proteins may be controlled by post-translational modifications that lead to structural changes such as folding, although this has not been observed. Here we show that multisite phosphorylation induces folding of the intrinsically disordered 4E-BP2, the major neural isoform of the family of three mammalian proteins that bind eIF4E and suppress cap-dependent translation initiation. In its non-phosphorylated state, 4E-BP2 interacts tightly with eIF4E using both a canonical YXXXXLΦ motif (starting at Y54) that undergoes a disorder-to-helix transition upon binding and a dynamic secondary binding site. We demonstrate that phosphorylation at T37 and T46 induces folding of residues P18–R62 of 4E-BP2 into a four-stranded β-domain that sequesters the helical YXXXXLΦ motif into a partly buried β-strand, blocking its accessibility to eIF4E. The folded state of pT37pT46 4E-BP2 is weakly stable, decreasing affinity by 100-fold and leading to an order-to-disorder transition upon binding to eIF4E, whereas fully phosphorylated 4E-BP2 is more stable, decreasing affinity by a factor of approximately 4,000. These results highlight stabilization of a phosphorylation-induced fold as the essential mechanism for phospho-regulation of the 4E-BP:eIF4E interaction and exemplify a new mode of biological regulation mediated by intrinsically disordered proteins.

Modulation of Intrinsically Disordered Protein Function by Post-translational Modifications

Post-translational modifications (PTMs) produce significant changes in the structural properties of intrinsically disordered proteins (IDPs) by affecting their energy landscapes. PTMs can induce a range of effects, from local stabilization or destabilization of transient secondary structure to global disorder-to-order transitions, potentially driving complete state changes between intrinsically disordered and folded states or dispersed monomeric and phase-separated states. Here, we discuss diverse biological processes that are dependent on PTM regulation of IDPs. We also present recent tools for generating homogenously modified IDPs for studies of PTM-mediated IDP regulatory mechanisms.

Rapid Kinetics of Na+ Binding to Thrombin

The kinetic mechanism of Na+ binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na+ binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose kobs decreases hyperbolically with increasing [Na+]. The rapid phase is due to Na+ binding to the enzyme E to generate the E:Na+ form. The slow phase is due to the interconversion between E* and E, where E* is a form that cannot bind Na+. Temperature studies in the range from 5 to 35 °C show significant enthalpy, entropy, and heat capacity changes associated with both Na+ binding and the E to E* transition. As a result, under conditions of physiologic temperature and salt concentrations, the E* form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na+ (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na+ binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na+ binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na+ binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.

Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4.

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 (38SFNGGPQNTFEEFPLSDIE56) and PAR4 (51KSSDKPNPR ↓ GYPGKFCANDSDTLELPASSQA81, ↓ = site of cleavage) have been solved at 2.0 and 3.5 Å resolution, respectively. The cleaved form of PAR3, traced in the electron density maps from Gln-44 to Glu-56, makes extensive hydrophobic and electrostatic contacts with exosite I of thrombin through the fragment 47FEEFPLSDIE56. Occupancy of exosite I by PAR3 allosterically changes the conformation of the 60-loop and shifts the position of Trp-60d ≈10 Å with a resulting widening of the access to the active site. The PAR4 fragment, traced entirely in the electron density maps except for five C-terminal residues, clamps Trp-60d, Tyr-60a, and the aryl-binding site of thrombin with Pro-56 and Pro-58 at the P2 and P4 positions and engages the primary specificity pocket with Arg-59. The fragment then leaves the active site with Gly-60 and folds into a short helical turn that directs the backbone away from exosite I and over the autolysis loop. The structures demonstrate that thrombin activation of PAR4 may occur with exosite I available to bind cofactor molecules, like the cleaved form of PAR3, whose function is to promote substrate diffusion into the active site by allosterically changing the conformation of the 60-loop.

Interaction of the Eukaryotic Initiation Factor 4E with 4E-BP2 at a Dynamic Bipartite Interface

Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs). Whereas the binding of 4E-BP peptides containing the eIF4E-binding ⁵⁴YXXXXLΦ⁶⁰ motif has been studied, atomic-level characterization of the interaction of eIF4E with full-length 4E-BPs has been lacking. Here, we use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to characterize the dynamic, structural and binding properties of 4E-BP2. Although disordered, 4E-BP2 contains significant fluctuating secondary structure and binds eIF4E at an extensive bipartite interface including the canonical ⁵⁴YXXXXLΦ⁶⁰ and ⁷⁸IPGVT⁸² sites. Each of the two binding elements individually has submicromolar affinity and exchange on and off of the eIF4E surface within the context of the overall nanomolar complex. This dynamic interaction facilitates exposure of regulatory phosphorylation sites within the complex. The 4E-BP2 interface on eIF4E overlaps yet is more extensive than the eIF4G:eIF4E interface, suggesting that these key interactions may be differentially targeted for therapeutics.

Crystal structure of thrombin in a self-inhibited conformation.

The activating effect of Na+ on thrombin is allosteric and depends on the conformational transition from a low activity Na+-free (slow) form to a high activity Na+-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na+ have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na+ site and the active site regions. Here, we report a 1.87Å resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow → fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10Å to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na+-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na+ and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.

Mutant N143P reveals how Na+ activates thrombin

The molecular mechanism of thrombin activation by Na+ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E, and E:Na+ forms. The extended scheme establishes that analysis of kcat unequivocally identifies allosteric transduction of Na+ binding into enhanced catalytic activity. The thrombin mutant N143P features no Na+-dependent enhancement of kcat yet binds Na+ with an affinity comparable to that of wild type. Crystal structures of the mutant in the presence and absence of Na+ confirm that Pro143 abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu192, which in turn controls the orientation of the Glu192-Gly193 peptide bond and the correct architecture of the oxyanion hole. We conclude that Na+ activates thrombin by securing the correct orientation of the Glu192-Gly193 peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143–192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na+ activation is present in all Na+-activated trypsin-like proteases.

Na(+) binding to meizothrombin desF1.

Abstract.Meizothrombin is the physiologically active intermediate generated by a single cleavage of prothrombin at R320 to separate the A and B chains. Recent evidence has suggested that meizothrombin, like thrombin, is a Na+-activated enzyme. In this study we present the first X-ray crystal structure of human meizothrombin desF1 solved in the presence of the active site inhibitor PPACK at 2.1 Å resolution. The structure reveals a Na+ binding site whose architecture is practically identical to that of human thrombin. Stopped-flow measurements of Na+ binding to meizothrombin desF1 document a slow phase of fluorescence change with a kobs decreasing hyperbolically with increasing [Na+], consistent with the existence of three conformations in equilibrium, E*, E and E:Na+, as for human thrombin. Evidence that meizothrombin exists in multiple conformations provides valuable new information for studies of the mechanism of prothrombin activation.

Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation

Significance The cell is divided into compartments where specific biochemical functions are performed. These compartments can be delineated by membranes or through phase separation of proteins or protein and nucleic acids to form membraneless organelles. The latter situation occurs with an intrinsically disordered region of Ddx4, a major constituent of germ granules. The nature of the interior of membraneless organelles is poorly understood. Here, we use NMR to show that the intrinsically disordered Ddx4 region remains disordered and highly dynamic in the phase-separated state, while diffusing as slowly as a particle the size of a bacterial cell. Ddx4 molecules form a network of interactions on phase separation, providing an alternative environment to that found in membrane-encapsulated organelles. Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid “membraneless organelles” that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, Ddx4cond, diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing Ddx4cond to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of Ddx4cond have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in Ddx4cond are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in Ddx4cond are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.