Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the α subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.

The fidelity of the poliovirus RNA-dependent RNA polymerase (3Dpol) plays a direct role in the genomic evolution and pathogenesis of the virus. A single site mutation (Gly64Ser) that is remote from the catalytic center results in a higher fidelity polymerase. NMR studies with [methyl-13C]methionine-labeled protein were used to compare the solution structure and dynamics of wild-type and Gly64Ser 3Dpol. The chemical shifts for the Met6 resonance were significantly different between wild-type and Gly64Ser 3Dpol when bound in ternary complexes with RNA and incorrect, but not with correct, nucleotide, suggesting that the Gly64Ser mutation induces structural changes in the N-terminal β-strand when the enzyme is bound to incorrect but not correct nucleotide. We also observe changes in the transverse relaxation times for methionines near regions important for nucleotide and RNA binding and catalysis. Our strategy to assign the [methyl-13C]methionine resonances involved separately mutating each of the 17 methionines. Several substitutions produced additional resonances for both Met6 and Met187, a reporter for RNA binding, and conformational changes in the highly conserved motif B loop, even though these methionines are greater than 20 Å apart. The results for Gly64Ser and the other mutants are intriguing considering that they can result in structural and/or dynamic changes to methionines distant from the site of mutation. We propose that there is a long-distance network operating throughout 3Dpol that coordinates ligand binding, conformational changes, and catalysis. Mutation of Gly64 results in structural and/or dynamic changes to the network that may affect polymerase fidelity.

Fast, accurate nucleotide incorporation by polymerases facilitates expression and maintenance of genomes. Many polymerases use conformational dynamics of a conserved α helix to permit efficient nucleotide addition only when the correct nucleotide substrate is bound. This α helix is missing in structures of RNA-dependent RNA polymerases (RdRps) and RTs. Here, we use solution-state nuclear magnetic resonance to demonstrate that the conformation of conserved structural motif D of an RdRp is linked to the nature (correct versus incorrect) of the bound nucleotide and the protonation state of a conserved, motif-D lysine. Structural data also reveal the inability of motif D to achieve its optimal conformation after incorporation of an incorrect nucleotide. Functional data are consistent with the conformational change of motif D becoming rate limiting during and after nucleotide misincorporation. We conclude that motif D of RdRps and, by inference, RTs is the functional equivalent to the fidelity helix of other polymerases.Highlights► Motif D changes conformation upon binding “correct,” but not “incorrect,” NTP ► Structural change in motif D depends on the protonation state of the motif D lysine ► The motif D lysine is also an important determinant of polymerase fidelity ► Motif D has an analogous function to helix O/P in A/B family DNA polymerases

•Binding to a viral protein influences structural dynamics at another binding site•Ligand binding also influences the function of the other binding site•Control of the energy landscape may regulate myriad functions of this viral protein•Protein structural dynamics effectively increases the genomic information contentThe 3C protein is a master regulator of the picornaviral infection cycle, responsible for both cleaving viral and host proteins, and interacting with genomic RNA replication elements. Here we use nuclear magnetic resonance spectroscopy and molecular dynamics simulations to show that 3C is conformationally dynamic across multiple timescales. Binding of peptide and RNA lead to structural dynamics changes at both the protease active site and the RNA-binding site, consistent with these sites being dynamically coupled. Indeed, binding of RNA influences protease activity, and likewise, interactions at the active site affect RNA binding. We propose that RNA and peptide binding re-shapes the conformational energy landscape of 3C to regulate subsequent functions, including formation of complexes with other viral proteins. The observed channeling of the 3C energy landscape may be important for regulation of the viral infection cycle.

The α-subunit of tryptophan synthase (αTS) catalyzes the conversion of indole-3-glycerol phosphate to d-glyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to αTS are modulated by the binding of the β-subunit to regulate αTS function. Understanding these long-range amino acid networks in αTS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of αTS throughout its catalytic cycle, in the absence of the β-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that αTS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the β2α2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the β2α2 and β6α6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale.Download high-res image (195KB)Download full-size imageHighlights► Allosteric networks in αTS regulate function. ► The phosphate-binding pocket and active-site loops in αTS are structurally coupled. ► Ligands induce long-range structural and dynamic changes in αTS. ► Allosteric pathways in αTS change during catalysis. ► β-Subunit may regulate αTS function through modulating allosteric networks.