A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction

BACKGROUND: Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS: Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS: The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.     

A mechanism for the evolution of phosphorylation sites

Protein phosphorylation provides a mechanism for the rapid, reversible control of protein function. Phosphorylation adds negative charge to amino acid side chains, and negatively charged amino acids (Asp/Glu) can sometimes mimic the phosphorylated state of a protein. Using a comparative genomics approach, we show that nature also employs this trick in reverse by evolving serine, threonine, and tyrosine phosphorylation sites from Asp/Glu residues. Structures of three proteins where phosphosites evolved from acidic residues (DNA topoisomerase II, enolase, and C-Raf) show that the relevant acidic residues are present in salt bridges with conserved basic residues, and that phosphorylation has the potential to conditionally restore the salt bridges. The evolution of phosphorylation sites from glutamate and aspartate provides a rationale for why phosphorylation sometimes activates proteins, and helps explain the origins of this important and complex process.     

1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.     

Coarse-grained dynamics of the receiver domain of NtrC: fluctuations, correlations and implications for allosteric cooperativity

Receiver domains are key molecular switches in bacterial signaling. Structural studies have shown that the receiver domain of the nitrogen regulatory protein C (NtrC) exists in a conformational equilibrium encompassing both inactive and active states, with phosphorylation of Asp54 allosterically shifting the equilibrium towards the active state. To analyze dynamical fluctuations and correlations in NtrC as it undergoes activation, we have applied a coarse-grained dynamics algorithm using elastic network models. Normal mode analysis reveals possible dynamical pathways for the transition of NtrC from the inactive state to the active state. The diagonalized correlation between the inactive and the active (phosphorylated) state shows that most correlated motions occur around the active site of Asp54 and in the region Thr82 to Tyr101. This indicates a coupled correlation of dynamics in the "Thr82-Tyr101" motion. With phosphorylation inducing significant flexibility changes around the active site and alpha3 and alpha4 helices, we find that this activation makes the active-site region and the loops of alpha3/beta4 and alpha4/beta5 more stable. This means that phosphorylation entropically favors the receiver domain in its active state, and the induced conformational changes occur in an allosteric manner. Analyses of the local flexibility and long-range correlated motion also suggest a dynamics criterion for determining the allosteric cooperativity of NtrC, and may be applicable to other proteins.     

Detailed regulatory mechanism of the interaction between ZO-1 PDZ2 and connexin43 revealed by MD simulations

The gap junction protein connexin43 (Cx43) binds to the second PDZ domain of Zonula occludens-1 (ZO-1) through its C-terminal tail, mediating the regulation of gap junction plaque size and dynamics. Biochemical study demonstrated that the very C-terminal 12 residues of Cx43 are necessary and sufficient for ZO-1 PDZ2 binding and phosphorylation at residues Ser (-9) and Ser (-10) of the peptide can disrupt the association. However, only a crystal structure of ZO-1 PDZ2 in complex with a shorter 9 aa peptide of connexin43 was solved experimentally. Here, the interactions between ZO-1 PDZ2 and the short, long and phosphorylated Cx43 peptides were studied using molecular dynamics (MD) simulations and free energy calculation. The short peptide bound to PDZ2 exhibits large structural variations, while the extension of three upstream residues stabilizes the peptide conformation and enhanced the interaction. Phosphorylation at Ser(-9) significantly weakens the binding and results in conformational flexibility of the peptide. Glu210 of ZO-1 PDZ2 was found to be a key regulatory point in Cx43 binding and phosphorylation induced dissociation.     

Dimer-induced signal propagation in Spo0A

Spo0A, the response regulator protein controlling the initiation of sporulation in Bacillus, has two distinct domains, an N-terminal phosphoacceptor (or receiver) domain and a C-terminal DNA-binding (or effector) domain. The phosphoacceptor domain mediates dimerization of Spo0A on phosphorylation. A comparison of the crystal structures of phosphorylated and unphosphorylated response regulators suggests a mechanism of activation in which structural changes originating at the phosphorylatable aspartate extend to the alpha4beta5alpha5 surface of the protein. In particular, the data show an important role in downstream signalling for a conserved aromatic residue (Phe-105 in Spo0A), the conformation of which alters upon phosphorylation. In this study, we have prepared a Phe-105 to Ala mutant to probe the contribution of this residue to Spo0A function. We have also made an alanine substitution of the neighbouring residue Tyr-104 that is absolutely conserved in the Spo0As of spore-forming Bacilli. The spo0A(Y104A) and spo0A(F105A) alleles severely impair sporulation in vivo. In vitro phosphorylation of the purified proteins by phosphoramidate is unaffected, but dimerization and DNA binding are abolished by the mutations. We have identified intragenic suppressor mutations of spo0A(F105A) and shown that these second-site mutations in the purified proteins restore phosphorylation-dependent dimer formation. Our data support a model in which dimerization and signal transduction between the two domains of Spo0A are mediated principally by the alpha4beta5alpha5 signalling surface in the receiver domain.     

Conformational sampling and polarization of Asp26 in pKa calculations of thioredoxin

Thioredoxin is a protein that has been used as model system by various computational methods to predict the pK_a of aspartate residue Asp26 which is 3.5 units higher than a solvent exposed one (eg, Asp20). Here, we use extensive atomistic molecular dynamics simulations of two different protonation states of Asp26 in combination with conformational analysis based on RMSD clustering and principle component analysis to identify representative conformations of the protein in solution. For each conformation, the Gibbs free energy of proton transfer between Asp26 and Asp20, which is fully solvated in a loop region of the protein, is calculated with the Amber99sb force field in alchemical transformations. The varying polarization of the two residues in different molecular environments and protonation states is described by Hirshfeld-I (HI) atomic charges obtained from the averaged polarized electron density. Our results show that the Gibbs free energy of proton transfer is dependent on the protein conformation, the proper sampling of the neighboring Lys57 residue orientations and on water molecules entering the hydrophobic cavity upon deprotonating Asp26. The inclusion of the polarization of both aspartate residues in the free energy cycle by HI atomic charges corrects the results from the non-polarizable force field and reproduces the experimental ΔpK_a value of Asp26.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Self‐guided Langevin dynamics study of regulatory interactions in NtrC

Multiple self‐guided Langevin dynamics (SGLD) simulations were performed to examine structural and dynamical properties of the receiver domain of nitrogen regulatory protein C (NtrC^r). SGLD and MD simulations of the phosphorylated active form structure suggest a mostly stable but broad structural ensemble of this protein. The finite difference Poisson–Boltzmann calculations of the pK_a values of the active site residues suggest an increase in the pK_a of His‐84 on phosphorylation of Asp‐54. In SGLD simulations of the phosphorylated active form with charged His‐84, the average position of the regulatory helix α4 is found closer to the starting structure than in simulations with the neutral His‐84. To model the transition pathway, the phosphate group was removed from the simulations. After 7 ns of simulations, the regulatory helix α4 was found approximately halfway between positions in the NMR structures of the active and inactive forms. Removal of the phosphate group stimulated loss of helix α4, suggesting that the pathway of conformational transition may involve partial unfolding mechanism. The study illustrates the potential utility of the SGLD method in studies of the coupling between ligand binding and conformational transitions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Distinctive solution conformation of phosphatase inhibitor CPI-17 substituted with aspartate at the phosphorylation-site threonine residue

We present solution NMR structures for wild-type and mutated forms of CPI-17, a phosphoinhibitor for protein phosphatase 1. Phosphorylation of Thr38 of CPI-17 produces a >1000-fold increase in inhibitory potency for myosin phosphatase. We compared the 1H-15N heteronuclear single quantum coherence spectroscopy (HSQC) chemical shifts of wild-type CPI-17, partially phosphorylated CPI-17 and CPI-17 with Thr38 replaced with Asp to introduce a negative charge. There was a switch in the protein conformation due to either Asp substitution or phosphorylation, so we determined the solution NMR structure of the CPI-17 T38D mutant as a model for the active (phospho-) conformation. The structures reveal a molecular switch in conformation that involves the rotation of two of the four helices in the four helix bundle. Despite this conformational switch, there was little increase in the inhibitory potency with T38D. We propose that for this inhibitor, a negative charge at residue 38 is sufficient to trigger an active conformation, but a phosphoryl group is required for full inhibitory potency against protein phosphatase-1.     

Conformational changes in protein loops and helices induced by post-translational phosphorylation

Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can “capture” a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins.     

Structural Analysis of Divalent Metals Binding to the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Response Regulator Spo0F: The Possibility for <Emphasis Type="Italic">In Vitro</Emphasis> Metalloregulation in the Initiation of Sporulation

The presence of a divalent metal ion in a negatively charged aspartic acid pocket is essential for phosphorylation of response regulator proteins. Here, we present metal binding studies of the Bacillus subtilis response regulator Spo0F using NMR and μESI-MS. NMR studies show that the divalent metals Ca²⁺, Mg²⁺ and Mn²⁺ primarily bind, as expected, in the Asp pocket phosphorylation site. However, identical studies with Cu²⁺ show distinct binding effects in three specific locations: (i) the Asp pocket, (ii) a grouping of charged residues at a site opposite of the Asp pocket, and (iii) on the β4-α4 loop and the β5/α5 interface, particularly around and including H101. μESI-MS studies stoichiometrically confirm the NMR studies and demonstrate that most divalent metal ions bind to Spo0F primarily in a 1:1 ratio. Again, in the case of Cu²⁺, multiple metal-bound species are observed. Subsequent experiments reveal that Mg²⁺ supports phosphotransfer between KinA and Spo0F, while Cu²⁺ fails to support KinA phosphotransfer. Additionally, the presence of Cu²⁺ at non-lethal concentrations in sporulation media for B. subtilis and the related organism Pasteuria penetrans was found to inhibit spore formation while continuing to permit vegetative growth. Depending on the type of divalent metal ion present, in vitro phosphorylation of Spo0F by its cognate kinase KinA can be inhibited.     

Phosphorylated aspartate in the structure of a response regulator protein

Phosphorylation of aspartic acid residues is the hallmark of two- component signal transduction systems that orchestrate the adaptive responses of micro-organisms to changes in their surroundings. Two-component systems consist of a sensor kinase that interprets environmental signals and a response regulator that activates the appropriate physiological response. Although structures of response regulators are known, little is understood about their activated phosphorylated forms, due to the intrinsic instability of the acid phosphate linkage. Here, we report the phosphorylated structure of the receiver/phosphoacceptor domain of Spo0A, the master regulator of sporulation, from Bacillus stearothermophilus. The phosphoryl group is covalently bonded to the invariant aspartate 55, and co-ordinated to a nearby divalent metal cation, with both species fulfilling their electrostatic potential through interactions with solvent water molecules, the protein main chain, and with side-chains of amino acid residues strongly conserved across the response regulator family. This is the first direct visualisation of a phosphoryl group covalently linked to an aspartic acid residue in any protein, with implications for signalling within the response regulator family.     

The crystal structure of beryllofluoride Spo0F in complex with the phosphotransferase Spo0B represents a phosphotransfer pretransition state

A number of regulatory circuits in biological systems function through the exchange of phosphoryl groups from one protein to another. Spo0F and Spo0B are components of a phosphorelay that control sporulation in the bacterium Bacillus subtilis through the exchange of a phosphoryl group. Using beryllofluoride as a mimic for phosphorylation, we trapped the interaction of the phosphorylated Spo0F with Spo0B in the crystal lattice. The transition state of phosphoryl transfer continues to be a highly debated issue, as to whether it is associative or dissociative in nature. The geometry of Spo0F binding to Spo0B favors an associative mechanism for phosphoryl transfer. In order to visualize the autophosphorylation of the histidine kinase, KinA, and the subsequent phosphoryl transfer to Spo0F, we generated in silico models representing these reaction steps.     

Molecular dynamics studies on structure and dynamics of phospholamban monomer and pentamer in membranes

Phospholamban (PLB) is an integral membrane protein of 52 residues that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle cells through reversible phosphorylation of Ser16. To explore its possible conformations and dynamics in a monomeric state, we have performed comparative molecular dynamics simulations of unphosphorylated and phosphorylated PLB (pPLB) with various orientations in POPC membranes. The simulations indicate that dynamics of the cytoplasmic domain is highly dependent on its interactions with membranes, that is, large conformational changes in the absence of membrane interactions, but very restricted dynamics in their presence. pPLB shows more structural flexibility in its cytoplasmic domain, which is consistent with experimental observations. We have also performed a simulation of a PLB pentameric structure (the so‐called bellflower model), recently determined in micelles, to investigate its behaviors in a POPC membrane. The cytoplasmic domain in each monomer shows uncorrelated dynamics and undergoes large conformational changes toward the membrane surface during the simulation, which supports the so‐called pinwheel model of the PLB pentamer structure. The hydrophobic nature of the pentameric pore excludes water molecules in the pore region, which illustrates that the pore appears to be an energetic barrier for ion and water translocation. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Phosphorylation of deinococcal RecA affects its structural and functional dynamics implicated for its roles in radioresistance of Deinococcus radiodurans

Abstract

Deinococcus RecA (DrRecA) protein is a key repair enzyme and contributes to efficient DNA repair of Deinococcus radiodurans. Phosphorylation of DrRecA at Y77 (tyrosine 77) and T318 (threonine 318) residues modifies the structural and conformational switching that impart the efficiency and activity of DrRecA. Dynamics comparisons of DrRecA with its phosphorylated analogues support the idea that phosphorylation of Y77 and T318 sites could change the dynamics and conformation plasticity of DrRecA. Furthermore, docking studies showed that phosphorylation increases the binding preference of DrRecA towards dATP versus ATP and for double-strand DNA versus single-strand DNA. This work supporting the idea that phosphorylation can modulate the crucial functions of this protein and having good concordance with the experimental data. Abbreviations DrRecA Deinococcus RecA

DSB DNA double-strand breaks

hDNA heteroduplex DNA

STYPK serine/threonine/tyrosine protein kinase

T318 threonine 318

Y77 tyrosine 77

Communicated by Ramaswamy H. Sarma