Michael K. Fenwick

Expanded ensemble and replica exchange methods for simulation of protein-like systems

Extended state methods are powerful tools for studying the conformational equilibria of proteins. This study focuses on three aspects of their implementation. First, existing approaches for determining importance weights (namely, recursion, random walk, and transition probability schemes) are compared in the context of their use with the method of expanded ensembles (EXE). Second, a combined scheme (REXE) involving EXE and replica exchange (REX) updates is developed for simulating a small number of replicas within a much larger macrostate space. Finally, variants of the extended state methods are considered for accelerating folding, either through special-purpose ensembles which target specific force-field parameters, or through biased sampling of extended macrostates that favor structural fluctuations. All methods are applied to a three-dimensional lattice protein model. Overall, it is found that transition probability approaches employing multiple system replicas perform naturally better than methods that intrinsically require macrostate equilibration by a single replica; the transition probability approaches need about an order of magnitude fewer steps to reach the same degree of convergence in the importance weights. The specific REXE protocol implemented is observed to have an efficiency intermediate to that of EXE and REX schemes at high temperatures, but to outperform them at more glassy conditions. Finally, special-purpose and locally enhanced tempering ensembles are shown to promote faster folding than conventional tempering.

Structure of Glutamate Receptors

Glutamate receptors mediate a vast array of processes in plants, animals and bacteria. In particular, the ionotropic glutamate receptors (iGluRs) are the most abundant excitatory neurotransmitter receptors in the mammalian central nervous system. Because these proteins are constructed from distinct folding domains, most of which can be traced to bacterial precursors, the analyses of these important receptor proteins has been performed on a variety of levels ranging from atomic structure and dynamics to behavioral studies. This review will focus on the structure and dynamics of iGluRs, with particular emphasis on the role that the glutamate-binding domain (S1S2) plays in receptor function.

Mechanism of partial agonism at the GluR2 AMPA receptor: Measurements of lobe orientation in solution

The mechanism by which the binding of a neurotransmitter to a receptor leads to channel opening is a central issue in molecular neurobiology. The structure of the agonist binding domain of ionotropic glutamate receptors has led to an improved understanding of the changes in structure that accompany agonist binding and have provided important clues about the link between these structural changes and channel activation and desensitization. However, because the binding domain has exhibited different structures under different crystallization conditions, understanding the structure in the absence of crystal packing is of considerable importance. The orientation of the two lobes of the binding domain in the presence of a full agonist, an antagonist, and several partial agonists was measured using NMR spectroscopy by employing residual dipolar couplings. For some partial agonists, the solution conformation differs from that observed in the crystal. A model of channel activation based on the results is discussed.

NMR spectroscopy of the ligand-binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists.

Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter-receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bilobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure-function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization, whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand-protein interactions and lobe closure that may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC(50) data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences.

Brownian dynamics simulation of the motion of a rigid sphere in a viscous fluid very near a wall

Total Internal Reflection Microscopy (TIRM) is an experimental technique that allows the potential energy profile of a colloidal particle above a plate to be determined from the particle’s observed Brownian motion. We have used Brownian Dynamics (BD) simulations to numerically simulate TIRM experiments. We show that a careful treatment of the position dependence of the colloidal particle’s mobility near the plate is vital to performing realistic simulations. These simulations enable us to systematically study the effect of experiment duration, data sampling methods, and physical parameters of the suspended colloid on the accuracy of potential energy profiles determined via TIRM. We also present an analytical theory to predict the range of energies reliably probed by TIRM experiments. This theory provides simple guidelines for the design and optimization of future TIRM experiments. Finally, we have used BD simulations to investigate the use of radiation pressure and the effects of experimental noise in TIR...

On the Mechanisms of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Binding to Glutamate and Kainate

The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediates much of the fast excitatory neurotransmission in the central nervous system. The ability of these receptors to shape such responses appears to be due in part to dynamic processes induced by agonists in the ligand-binding domain. Previous studies employing fluorescence spectroscopy and whole cell recording suggest that agonist binding is followed by sequential transitions to one or more distinct conformational states. Here, we used hydrogen-deuterium exchange to determine the mechanisms of binding of glutamate and kainate (full and partial agonists, respectively) to a soluble ligand-binding domain of GluR2. Our results provide a structural basis for sequential state models of agonist binding and the free energy changes of the associated state-to-state transitions. For glutamate, a multi-equilibrium binding reaction was discerned involving distinct ligand docking, domain isomerization, and lobe-locking steps. In contrast, kainate binding involves a simpler dock-isomerization process in which the isomerization equilibrium is shifted dramatically toward open domain conformations. In light of increasing evidence that the stability, in addition to the extent, of domain closure is a critical component of the channel activation mechanism, the differences in domain opening and closing equilibria detected for glutamate and kainate should be useful structural measures for interpreting the markedly different current responses evoked by these agonists.

Dynamics of Cleft Closure of the GluA2 Ligand Binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

Background: Glutamate receptors are essential proteins for transmitting information in the CNS. Results: The stability of H-bonds at multiple points within the ligand-binding domain varies with the efficacy of the bound agonist. Conclusion: H-bonds inside and outside of the binding pocket contribute to channel activation and desensitization. Significance: Fine-tuning of glutamate receptor responses is dependent upon electrostatic interactions and H-bonds outside of the binding pocket. The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.

Thiamin pyrimidine biosynthesis in Candida albicans : a remarkable reaction between histidine and pyridoxal phosphate.

In Saccharomyces cerevisiae , thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.

Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5′-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

Structural studies of viperin, an antiviral radical SAM enzyme.

Significance We report structures of viperin, an antiviral radical S-adenosylmethionine (SAM) enzyme. The overall structure shows a canonical radical SAM enzyme fold that harbors a [4Fe-4S] cluster. Structures with a bound SAM analog or SAM cleavage products are consistent with a conventional mechanism of radical formation. Sequence alignments guided by the putative active site residues of viperin reveal viperin-like enzymes in species from all kingdoms of life. Structural alignments show similarity between viperin and the molybdenum cofactor biosynthetic enzyme MoaA and show that the active site architecture of viperin is consistent with a nucleoside triphosphate substrate. Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45–362) complexed with S-adenosylhomocysteine (SAH) or 5′-deoxyadenosine (5′-dAdo) and l-methionine (l-Met). Viperin contains a partial (βα)6-barrel fold with a disordered N-terminal extension (residues 45–74) and a partially ordered C-terminal extension (residues 285–362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5′-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5′-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.