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The calmodulin (CaM) activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2 K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF-2 K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution-state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca2+ -CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C-lobe surface, complementary to the one involved in recognizing the calmodulin-binding domain (CBD) of TR, provides a secondary TR-interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca2+ -CaM•TR complex. Solution NMR data suggest that CaMH107K , which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaMH107K to induce TR autophosphorylation compared to wild-type CaM. This article is protected by copyright. All rights reserved.

BY-kinases (for bacterial tyrosine kinases) constitute a family of protein tyrosine kinases that are highly conserved in the bacterial kingdom and occur most commonly as essential components of multicomponent assemblies responsible for the biosynthesis, polymerization, and export of complex polysaccharides involved in biofilm or capsule formation. BY-kinase function has been attributed to a cyclic process involving formation of an oligomeric species, its disassembly into constituent monomers, and subsequent reassembly, depending on the overall phosphorylation level of a C-terminal cluster of tyrosine residues. However, the relationship of this process to the active/inactive states of the enzyme and the mechanism of its integration into the polysaccharide production machinery remain unclear. Here, we synthesize the substantial body of biochemical, cell-biological, structural, and computational data, acquired over the nearly 3 decades since the discovery of BY-kinases, to suggest means by which they fulfill their physiological function. We propose a mechanism involving temporal coordination of the assembly/disassembly process with the autokinase activity of the enzyme and its ability to be dephosphorylated by its counteracting phosphatase. We speculate that this temporal control enables BY-kinases to function as molecular timers that coordinate the diverse processes involved in the synthesis, polymerization, and export of complex sugar derivatives. We suggest that BY-kinases, which deploy distinctive catalytic domains resembling P-loop nucleoside triphosphatases, have uniquely adapted this ancient fold to drive functional processes through exquisite spatiotemporal control over protein-protein interactions and conformational changes. It is our hope that the hypotheses proposed here will facilitate future experiments targeting these unique protein kinases.

Eukaryotic elongation factor 2 kinase (eEF‐2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF‐2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF‐2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM‐regulated kinases. We had previously identified a minimal construct of eEF‐2K (TR) that is activated similarly to the wild‐type enzyme by CaM in vitro and retains its ability to phosphorylate eEF‐2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1‐methyls of TR and Ile δ1‐ and Met ε‐methyls of CaM, as probes of their mutual interaction and the influence of Ca(2+) thereon. We find that in the absence of Ca(2+), CaM exclusively utilizes its C‐terminal lobe (CaM(C)) to engage the N‐terminal CaM‐binding domain (CBD) of TR in a high‐affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca(2+)‐loaded N‐terminal lobe of CaM (CaM(N)) at increased Ca(2+) levels serves to enhance the affinity further. These latter interactions under Ca(2+) saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca(2+)‐free state. Our data are consistent with a scenario in which Ca(2+) enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM‐bound species without significantly modifying the conformation of TR within the final, active complex.

Bacterial tyrosine kinases (BY-kinases) and shikimate kinases (SKs) comprise two structurally divergent P-loop containing enzyme families that share similar catalytic site geometries, most notably with respect to their Walker-A, Walker-B, and DxD motifs. We had previously demonstrated that in BY-kinases, a specific interaction between the Walker-A and Walker-B motifs, driven by the conserved “catalytic” lysine housed on the former, leads to a conformation that is unable to efficiently coordinate Mg2+•ATP and is therefore incapable of chemistry. Here, using enhanced sampling molecular dynamics simulations, we demonstrate that structurally similar interactions between the Walker-A and Walker-B motifs, also mediated by the catalytic lysine, stabilize a state in SKs that deviates significantly from one that is necessary for the optimal coordination of Mg2+•ATP. This structural role of the Walker-A lysine is a general feature in SKs and is found to be present in members that encode a Walker-B sequence characteristic of the family (Coxiella burnetii SK), and in those that do not (Mycobacterium tuberculosis SK). Thus, the structural role of the Walker-A lysine in stabilizing an inactive state, distinct from its catalytic function, is conserved between two distantly related P-loop containing kinase families, the SKs and the BY-kinases. The universal conservation of this element, and of the key characteristics of its associated interaction partners within the Walker motifs of P-loop containing enzymes, suggests that this structural role of the Walker-A lysine is perhaps a widely deployed regulatory mechanism within this ancient family.

Eukaryotic elongation factor 2 kinase (eEF-2K), an atypical calmodulin-activated protein kinase, regulates translational elongation by phosphorylating its substrate, eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome. The activation and activity of eEF-2K are critical for survival under energy-deprived conditions and is implicated in a variety of essential physiological processes. Previous biochemical experiments have indicated that the binding site for the substrate eEF-2 is located in the C-terminal domain of eEF-2K, a region predicted to harbor several α-helical repeats. Here, using NMR methodology we have determined the solution structure of a C-terminal fragment of eEF-2K, eEF-2K(562–725) that encodes two α-helical repeats. The structure of eEF-2K(562–725) shows signatures characteristic of TPR domains and of their SEL1-like sub-family. Further, using the analyses of NMR spectral perturbations and ITC measurements, we have localized the eEF-2 binding site on eEF-2K(562–725). We find that eEF-2K(562–725) engages eEF-2 with an affinity comparable to that of the full-length enzyme. Further, eEF-2K(562–725) is able to inhibit the phosphorylation of eEF-2 by full-length eEF-2K in trans. Our present studies establish that eEF-2K(562–725) encodes the major elements necessary to enable the eEF-2K/eEF-2 interactions.

Extracellular signal-regulated kinase 1/2 (ERK1/2) constitute a point of convergence for complex signaling events that regulate essential cellular processes, including proliferation and survival. As such, dysregulation of the ERK signaling pathway is prevalent in many cancers. In the case of BRAF-V600E mutant melanoma, ERK inhibition has emerged as a viable clinical approach to abrogate signaling through the ERK pathway, even in cases where MEK and Raf inhibitor treatments fail to induce tumor regression due to resistance mechanisms. Several ERK inhibitors that target the active site of ERK have reached clinical trials, however, many critical ERK interactions occur at other potentially druggable sites on the protein. Here we discuss the role of ERK signaling in cell fate, in driving melanoma, and in resistance mechanisms to current BRAF-V600E melanoma treatments. We explore targeting ERK via a distinct site of protein-protein interaction, known as the D-recruitment site (DRS), as an alternative or supplementary mode of ERK pathway inhibition in BRAF-V600E melanoma. Targeting the DRS with inhibitors in melanoma has the potential to not only disrupt the catalytic apparatus of ERK but also its noncatalytic functions, which have significant impacts on spatiotemporal signaling dynamics and cell fate.

Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum. Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog–bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive “mixed” intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.

Measurement of nuclear spin relaxation provides a powerful approach to access information about biomolecular conformational dynamics over several orders of magnitude in timescale. In several cases this knowledge in combination with spatial information from three-dimensional structures yields unique insight into protein stability and the kinetics and thermodynamics of their interactions and function. However, due to intrinsic difficulties in studying large systems using solution state nuclear magnetic resonance (NMR) approaches, until recently these measurements were limited to small-to-medium-sized systems. However, the development of a wide range of novel strategies that allow the selective isotope labeling of methyl groups in proteins have allowed the exploitation of the unique relaxation properties of this spin-system. This has in turn enabled the extension of NMR approaches to high molecular weight proteins including a variety of enzymes and their complexes. Here, we recount our experiences in obtaining assignments of the methyl resonances for two representative members of a class of RNA-directed RNA polymerases (RdRps) encoded by bacteriophages of the Cystoviridae family. We demonstrate the utility of these methyl probes, limited in number for one case and more numerous for the other, to investigate the conformational dynamics of RdRps on the fast (ps-ns) and slow (μs-ms) timescales.

Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM) activated α–kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca(2+) ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry (XLMS) and small angle X-ray scattering (SAXS) measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen deuterium exchange mass spectrometry (HXMS) and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.

Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5’-(T/C)CCTT↓, leading to the formation of a covalent DNA-(3’-phosphotyrosyl(274))-enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81-314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with lower cleavage site affinity compared to the full-length enzyme. The N-terminal domain (amino acids 1-80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ~2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution NMR methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB are underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength.

Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM)-dependent member of the unique α-kinase family, impedes protein synthesis by phosphorylating eEF-2. We recently identified Thr-348 and Ser-500 as two key autophosphorylation sites within eEF-2K that regulate its activity. eEF-2K is regulated by Ca2+ ions and multiple upstream signaling pathways, but how it integrates these signals into a coherent output, i.e. phosphorylation of eEF-2, is unclear. This study focuses on understanding how the post-translational phosphorylation of Ser-500 integrates with Ca2+ and CaM to regulate eEF-2K. CaM is shown to be absolutely necessary for efficient activity of eEF-2K, and Ca2+ is shown to enhance the affinity of CaM toward eEF-2K. Ser-500 is found to undergo autophosphorylation in cells treated with ionomycin and is likely also targeted by PKA. In vitro, autophosphorylation of Ser-500 is found to require Ca2+ and CaM and is inhibited by mutations that compromise binding of phosphorylated Thr-348 to an allosteric binding pocket on the kinase domain. A phosphomimetic Ser-500 to aspartic acid mutation (eEF-2K S500D) enhances the rate of activation (Thr-348 autophosphorylation) by 6-fold and lowers the EC50 for Ca2+/CaM binding to activated eEF-2K (Thr-348 phosphorylated) by 20-fold. This is predicted to result in an elevation of the cellular fraction of active eEF-2K. In support of this mechanism, eEF-2K knock-out MCF10A cells reconstituted with eEF-2K S500D display relatively high levels of phospho-eEF-2 under basal conditions. This study reports how phosphorylation of a regulatory site (Ser-500) integrates with Ca2+ and CaM to influence eEF-2K activity.

In spite of the availability of a significant amount of structural detail on docking interactions involving mitogen-activated protein kinases (MAPKs) and their substrates, the mechanism by which the disordered phospho-acceptor on the substrate transiently interacts with the kinase catalytic elements and is phosphorylated, often with high efficiency, remains poorly understood. Here, this dynamic interaction is analyzed in the context of available biophysical and biochemical data for ERK2, an archetypal MAPK. A hypothesis about the nature of the ternary complex involving a MAPK, its substrate, and ATP immediately prior to the chemical step (the pre-chemistry complex) is proposed. It is postulated that the solution ensemble (the pre-chemistry ensemble) representing the pre-chemistry complex comprises several conformations that are linked by dynamics on multiple timescales. These individual conformations possess different intrinsic abilities to proceed through the chemical step. The overall rate of chemistry is therefore related to the microscopic nature of the pre-chemistry ensemble, its constituent conformational microstates, and their intrinsic abilities to yield a phosphorylated product. While characterizing these microstates within the pre-chemistry ensemble in atomic or near-atomic detail is an extremely challenging proposition, recent developments in hybrid methodologies that employ computational approaches driven by experimental data appear to provide the most promising path forward toward achieving this goal.

P2, an RNA-directed RNA polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across the Cystoviridae family. These range from atomic resolution snapshots of P2 trapped in functionally significant states, in complex with catalytic/structural metal ions, polynucleotide templates and substrate nucleoside triphosphates, to P2 in the context of viral capsids providing structural insight into the assembly of supramolecular complexes and regulatory interactions therein. They include in vitro biochemical studies using P2 purified to homogeneity and in vivo studies utilizing infectious core particles. Recent advances in experimental techniques have also allowed access to the temporal dimension and enabled the characterization of dynamics of P2 on the sub-nanosecond to millisecond timescale through measurements of nuclear spin relaxation in solution and single molecule studies of transcription from seconds to minutes. Below we summarize the most significant results that provide critical insight into the role of P2 in regulating RNA synthesis in cystoviruses.

We have determined the structure of P2, the self-priming RdRp from cystovirus φ12 in two crystal forms (A, B) at resolutions of 1.7 A and 2.1 A. Form A contains Mg(2+) bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts-mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo.

In this issue of the Journal of Molecular Biology Xie et. al. describe the interaction between IL-2 inducible T-cell kinase (Itk), a non-receptor tyrosine kinase, and its substrate PLCγ1. The authors demonstrate that a previously determined set of basic amino acid residues1 on the C-terminal SH2 domain of PLCγ1, interacts with an acidic patch on the G helix (αG) of the catalytic domain of Itk. The results described here reinforce the importance of αG as a mediator of intermolecular interactions in protein kinases.

Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions. eEF-2KCBD engages CaM largely through the C lobe of the latter in an anti-parallel 1-5-8 hydrophobic mode reinforced by a pair of unique electrostatic contacts. Sparse interactions of eEF-2KCBD with the CaM N lobe results in persisting inter-lobe mobility. A conserved eEF-2K residue (W85) anchors it to CaM by inserting into a deep hydrophobic cavity within the CaM C lobe. Mutation of this residue (W85S) substantially weakens interactions between full-length eEF-2K and CaM in vitro and reduces eEF-2 phosphorylation in cells.

Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues. Our results, which represent the most detailed experimental characterization of fast dynamics in a viral RdRp until date, reveal a highly connected dynamic network as predicted by MD simulations of related systems. Our results suggest that the entry portals for template RNA and substrate NTPs are relatively disordered, while conserved motifs involved in metal binding, nucleotide selection, and catalysis display greater rigidity. Perturbations at the active site through metal binding or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with the limited experimental and extensive functional and in silico results available for homologous systems suggests conservation of the overall pattern of dynamics in viral RdRps.

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-kappaB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha2-alpha3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha2-alpha3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha2-alpha3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.

Detailed knowledge of the pH-dependence of ionizable residues in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein folding and stability. The reassembly of E. coli Thioredoxin (Trx) by complementation of its two disordered fragments (1-37/38-108) provides a folded heterodimer in equilibrium with its unfolded state which, based on circular dichroism and NMR spectroscopy, consists of two unfolded monomers. To gain insight into the role of electrostatics in protein folding and stability, we compared the pH-dependence of the carboxylate sidechain chemical shift of each Asp/Glu against that of its backbone amide chemical shift in the unfolded heterodimer. We monitored via C(CO)NH experiments four Asp and four Glu in fragments 38 to 108 (C37) of Trx in the pH range from 2.0 to 7.0 and compared them with results from (1)H(15)N HSQC experiments [Pujato et al., Biophys. J., 89 (2005) 3293-3302]. The (1)H(15)N HSQC analysis indicates two segments with quite distinct behavior: (A) a segment from Ala57 to Ala108 in which ionizable residues have up to three contiguous neighbors with pH-dependent backbone amide shifts, and (B) a segment of fifteen contiguous pH-dependent backbone amide shifts (Leu42 to Val56) in which two Asp and two Glu are implicated in medium range interactions. In all cases, the titration curves are simple modified sigmoidals from which a pH-midpoint (pH(m)) can be obtained by fitting. In segment A, the pH(m) of a given backbone amide of Asp/Glu mirrors within 0.15 pH-units that of its carboxylate sidechain (i.e., the pK(a)). In contrast, segment B shows significant differences with absolute values of 0.46 and 0.74 pH-units for Asp and Glu, respectively. The dispersion in the pH(m) of the backbone amide of Asp/Glu is also different in the two segments. Segment A shows a dispersion of 0.31 and 0.17 pH-units for Asp and Glu, respectively. Segment B shows a substantially larger dispersion (0.50 and 1.08 pH-units for Asp and Glu, respectively). In both segments, the dispersion in the pH(m) of its backbone amide is larger than in the pK(a) of the carboxylate sidechain (the latter is only 0.17 and 0.52 pH-units for Asp and Glu, respectively). Our results indicate that the pH(m) of the backbone amide chemical shift of Asp/Glu in a disordered polypeptide segment is a good predictor of its pK(a) whenever there are none or few neighboring backbone amides with similar pH-dependence.

We present here a stable isotope labeling technique for proteins, which seeks the appropriate compromise between the advantages of (a) random isotope labeling, with its large number of protons available for structure determination, and (b) selective labeling to generate isolated proton spins decreasing spectral complexity and improving relaxation properties of NMR experiments. The described reduced proton (REDPRO) procedure results in side-chain specific protonation of overexpressed proteins, which is highly selective. The REDPRO labeling scheme provides a sufficient number of NOE constraints for structure calculation. Dramatically improved relaxation properties of the heteronuclear magnetization transfer coupled with TROSY advantages make the proposed labeling scheme an attractive approach for study of high molecular weight protein targets, their ligand sites, and interdomain interactions.

In bacteriophages of the cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2. Here, using solution NMR techniques and the P2 and P7 proteins from cystovirus ϕ12, we demonstrate their largely electrostatic interaction in vitro. Chemical shift perturbations on P7 in the presence of P2 suggest that this interaction involves the dynamic C-terminal tail of P7, more specifically an acidic cluster therein. Patterns of chemical shift changes induced on P2 by the P7 C-terminus resemble those seen in the presence of single-stranded RNA suggesting similarities in binding. This association between P2 and P7 reduces the affinity of the former towards template RNA and results in its decreased activity both in de novo RNA synthesis and in extending a short primer. Given the presence of C-terminal acidic tracts on all cystoviral P7 proteins, the electrostatic nature of the P2/P7 interaction is likely conserved within the family and could constitute a mechanism through which P7 regulates transcription in cystoviruses.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.

International audience; The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (WzcCD). WzcCD binds in a largely YC-independent fashion near the Wzb catalytic site, inducing allosteric changes therein. YC dephosphorylation is proximity-mediated and reliant on the elevated concentration of phosphorylated YC near the Wzb active site resulting from WzcCD docking. Wzb principally recognizes the phosphate of its phosphotyrosine substrate and further stabilizes the tyrosine moiety through ring stacking interactions with a conserved active site tyrosine.

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone 15 N spin relaxation and 15 N, 1 H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.

Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymerase complex (PX) to replicate and transcribe the viral genome. Although the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior is understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Toward obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus ϕ12 at a resolution of 1.8 A. The N-terminal core region (1–129) of P7 forms a novel homodimeric α/β-fold having structural similarities with BRCT domains implicated in multiple protein–protein interactions in DNA repair proteins. Our results, combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly, hint toward its participation in key protein–protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130–169) that is essential for virus viability, although highly disordered, contains a nascent helix. We demonstrate for the first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminal tail of P7 play a role in RNA recognition. This interaction leads to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7–RNA interactions.

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.

0022-2836/$ see front matter © 2009 E Two errors appeared in this article. The abbreviation “SLS” was incorrectly defined as “strain layer superlattice” in the Abbreviations on page 402 and in the Materials and Methods section. The correct definition of “SLS” is static light scattering. On page 418, the statement “...followed by a DAWN EOS 14angle...“ should read “...followed by a DAWN EOS 18-angle...”

Heteronuclear NMR methods have been used to elucidate the secondary structure and the general tertiary fold of the protein NodF from Rhizobium leguminosarum. A similarity to acyl carrier proteins of the fatty acid synthase system had been suggested by the presence of a phosphopantetheine prosthetic group and a short stretch of sequence homology near the prosthetic group attachment site. NMR results suggest that the structural homology extends well beyond this region. Both proteins have three well-formed helices which fold in a parallel-antiparallel fashion and a prosthetic group attachment site near the beginning of the second helix.

A review is given of the mathematical foundations of average Liouvillian theory and examples are provided of average Liouvillian expressions derived for sequences in nuclear magnetic resonance which possess temporal symmetry or antisymmetry. The utility of this approach is demonstrated in the design of pulse sequences to measure the effect of cross-correlation between chemical shift anisotropy and dipolar couplings on relaxation rates in biomolecules. Experimental verification of the theory is carried out on a 15N labelled sample of ubiquitin.