The structural basis of cyclic diguanylate signal transduction by PilZ domains

The second messenger cyclic diguanylate (c-di-GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c-di-GMP and allosterically modulate effector pathways. We have determined a 1.9 A crystal structure of c-di-GMP bound to VCA0042/PlzD, a PilZ domain-containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain-containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C-terminal PilZ domain plus an N-terminal domain with a similar beta-barrel fold. C-di-GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven-residue long N-terminal loop that undergoes a conformational switch as it wraps around c-di-GMP. This switch brings the PilZ domain into close apposition with the N-terminal domain, forming a new allosteric interaction surface that spans these domains and the c-di-GMP at their interface. The very small size of the N-terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.     

Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.     

A structural comparison of ‘real’ and ‘model’ calmodulin clarified allosteric interactions regulating domain motion

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates various biochemical processes. CaM acts via structural changes and complex forming with its target enzymes. CaM has two globular domains (N-lobe and C-lobe) connected by a long linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, after that, entire CaM wraps the target enzyme through a large conformational change. However, the regulation mechanism, such as allosteric interactions regulating the conformational changes, is still unclear. In order to clarify the allosteric interactions, in this study, experimentally obtained ‘real’ structures are compared to ‘model’ structures lacking the allosteric interactions. As the allosteric interactions would be absent in calcium-free CaM (apo-CaM), allostery-eliminated calcium-bound CaM (holo-CaM) models were constructed by combining the apo-CaM’s linker and the holo-CaM’s N- and C-lobe. Before the comparison, the ‘real’ and ‘model’ structures were clustered and cluster–cluster relationship was determined by a principal component analysis. The structures were compared based on the relationship, then, a distance map and a contact probability analysis clarified that the inter-domain motion is regulated by several groups of inter-domain contacting residue pairs. The analyses suggested that these residues cause inter-domain translation and rotation, and as a consequence, the motion encourage structural diversity. The resultant diversity would contribute to the functional versatility of CaM.     

Cloning,expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.     

NMR investigations of allosteric processes in a two-domain Thermus thermophilus Hsp70 molecular chaperone

Hsp70 chaperones are two-domain proteins that assist in intra-cellular protein (re) folding processes in all species. The protein folding activity of the substrate binding domain of the Hsp70s is regulated by nucleotide binding at the nucleotide-binding domain through an as yet undefined heterotropic allosteric mechanism. The available structures of the isolated domains of Hsp70s have given very limited indications of nucleotide-induced conformational changes that could modulate the affinity for substrate proteins. Here, we present a multi-dimensional NMR study of a prokaryotic Hsp70 homolog, Thermus thermophilus DnaK, using a 54kDa construct containing both nucleotide binding domain and most of the substrate binding domain. It is determined that the nucleotide binding domain and substrate binding domain are closely associated in all ligand states studied. Comparison of the assigned NMR spectra of the two-domain construct with those of the previously studied isolated nucleotide binding domain, allowed the identification of the nucleotide binding domain-substrate binding domain interface. A global three-dimensional structure was obtained for the two-domain construct on the basis of this information and of NMR residual dipolar couplings measurements. This is the first experimental elucidation of the relative positioning of the nucleotide binding domain and substrate binding domain for any Hsp70 chaperone. Comparisons of NMR data between various ligand states including nucleotide-free, ATP, ADP.Pi and ADP.Pi+ peptide bound, identified residues involved in the allosteric inter-domain communication. In particular, peptide binding to the substrate binding domain was found to cause conformational changes in the NBD extending to the nucleotide binding pocket. Detailed analysis suggests that the inter-domain interface becomes tighter in the (nucleotide binding domain ligation/substrate binding domain ligation) order ATP/apo, ADP.Pi/apo ADP.Pi/peptide.     

Heterosubunit composition and crystal structures of a novel bacterial M16B metallopeptidase

Three subfamilies of metallopeptidase family M16 enzymes—M16A, M16B, and M16C—are widely distributed among eukaryotes and prokaryotes. SPH2681, a periplasmic M16B protein found in Sphingomonas sp. strain A1, contains an HXXEH motif essential for Zn²⁺ binding and catalytic activity. SPH2682 is another member of M16B, which lacks the metal-binding motif but conserves an active-site R/Y pair commonly found in the C-terminal half of M16 enzymes. Two genes coding for SPH2681 and SPH2682 assemble into a single operon in the bacterial genome. This study determined SPH2681 to be constitutively expressed in strain A1 cells grown on different carbon sources, suggesting a more general cellular function. SPH2681 and SPH2681/SPH2682 were overexpressed in Escherichia coli, purified, and characterized. SPH2681 was found to associate with SPH2682, forming a heterosubunit enzyme with peptidase activity, while SPH2681 alone exhibited no enzymatic activity. X-ray crystallography of the SPH2681/SPH2682 complex revealed two conformations (open and closed heterodimeric forms) within the same crystal. Compared with the closed form, the open form contains two subunits rotated away from each other by approximately 8°, increasing the distance between the zinc ion and active-site residues by up to 8 Å. In addition, many hydrogen bonds are formed or broken on change between the conformations of the heterodimers, suggesting that subunit dynamics is a prerequisite for catalysis. To our knowledge, this is the first report on both conformational forms of the same M16 peptidase, providing a unique insight into the general proteolytic mechanism of M16 proteases.     

Dynamic Allostery Controls Coat Protein Conformer Switching during MS2 Phage Assembly

Previously, an RNA stem-loop (TR) encompassing 19 nt of the genome of bacteriophage MS2 was shown to act as an allosteric effector of conformational switching in the coat protein during in vitro capsid assembly. TR RNA binding to symmetric coat protein dimers results in conformational changes, principally at the FG-loop connecting the F and G β-strands in each subunit, yielding an asymmetric structure. The FG-loops define the quasi-equivalent conformers of the coat protein subunit (A, B, and C) in the T = 3 capsid. Efficient assembly of this capsid in vitro requires that both symmetrical and asymmetrical forms of the coat protein dimer be present in solution, implying that they closely resemble the quasi-equivalent dimers (A/B and C/C) seen in the final capsid. Experiments show that assembly can be triggered by a number of RNA stem-loops unrelated to TR in sequence and detailed secondary structure, suggesting that there is little sequence specificity to the allosteric effect. Since the stem-loop binding site on the coat protein dimer is distal to the FG-loops the mechanism of this switching effect needs to be investigated. We have analyzed the vibrational modes of both TR-bound and RNA-free coat protein dimers using an all-atom normal-mode analysis. The results suggest that asymmetric contacts between the A-duplex RNA phosphodiester backbone and the EF-loop in one coat protein subunit result in the FG-loop of that subunit becoming more dynamic, whilst the equivalent loop on the other monomer decreases its mobility. The increased dynamic behaviour occurs in the FG-loop of the subunit required to undergo the largest conformational change when adopting the quasi-equivalent B conformation. The free energy barrier on the pathway to form this new structure would consequently be reduced compared to the unbound subunit. Our results also imply that the allosteric effect should be independent of the base sequence of the bound stem-loop, as observed experimentally. As a test of this model, we also examined the vibrational modes of a known assembly mutant, W82R, which cannot assemble beyond dimer. This mutation leads to an increased mobility of the DE-loop rather than the FG-loop after TR binding, consistent with the non-assembling phenotype of this mutant protein.     

Triggering loops and enzyme function: identification of loops that trigger and modulate movements

Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. beta1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.     

Structures of the “open” and “closed” state of trypanosomal triosephosphate isomerase,as observed in a new crystal form: Implications for the reaction mechanism

The structure of trypanosomal triosephosphate isomerase (TIM)has been solved at a resolution of 2.1Å in a new crystal form grown at pH 8.8 from PEG6000. In this new crystal form (space group C2, cell dimensions 94.8 Å, 48.3 Å, 131.0 Å, 90.0°, 100.3°, 90.0°), TIM is present in a ligand-free state. The asymmetric unit consists of two TIM subunits. Each of these subunits is part of a dimer which is sitting on a crystallographic twofold axis, such that the crystal packing is formed from two TIM dimers in two distinct environments. The two constituent monomers of a given dimer are, therefore, crystallographically equivalent. In the ligand-free state of TIM in this crystal form, the two types of dimer are very similar in structure, with the flexible loops in the “Open” conformation. For one dimer (termed molecule-1), the flexible loop (loop-6) is involved in crystal contacts. Crystals of this type have been used in soaking experiments with 0.4 M ammonium sulphate (studied at 2.4 Å resolution), and with 40 μM phosphoglycolohydroxamate (studied at 2.5 Å resolution). It is found that transfer to 0.4 M ammonuum sulphate (equal to 80 times the Ki of sulphate for TIM), gives rise to significant sulphate binding at the active site of one dimer (termed molecule-2), and less significant binding at the active site of the other. In neither dimer does sulphate induce a “closed” conformation. In a mother liquor containing 40 μM phosphoglycolohydroxamate (equal to 10 times the Ki of phosphoglycolohydroxamate for TIM), an inhibitor molecule binds at the active site of only that dimer of which the flexible loop is free from crystal contacts (molecule-2). In this dimer, it induces a closed conformation. These three structures are compared and discussed with respect to the mode of binding of ligand in the active site as well as with respect to the conformational changes resulting from ligand binding. © 1993 Wiley-Liss, Inc.     

Expression, Purification, Characterization, and X-Ray Analysis of Selenomethionine 215 Variant of Leukocyte Collagenase

Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et al. (1993) FEBS331, 134–140; Stöcker et al. (1995) Protein Sci.4, 823–840]. The distal S–CH₃ moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80–Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by Selenomethionine. Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry. The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in changed kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon urea denaturation. The X-ray crystal structure of this Selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.     

Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: nucleotide-free and nucleotide-bound conformations

The ABC-ATPase GlcV energizes a binding protein-dependent ABC transporter that mediates glucose uptake in Sulfolobus solfataricus. Here, we report high-resolution crystal structures of GlcV in different states along its catalytic cycle: distinct monomeric nucleotide-free states and monomeric complexes with ADP-Mg(2+) as a product-bound state, and with AMPPNP-Mg(2+) as an ATP-like bound state. The structure of GlcV consists of a typical ABC-ATPase domain, comprising two subdomains, connected by a linker region to a C-terminal domain of unknown function. Comparisons of the nucleotide-free and nucleotide-bound structures of GlcV reveal re-orientations of the ABCalpha subdomain and the C-terminal domain relative to the ABCalpha/beta subdomain, and switch-like rearrangements in the P-loop and Q-loop regions. Additionally, large conformational differences are observed between the GlcV structures and those of other ABC-ATPases, further emphasizing the inherent flexibility of these proteins. Notably, a comparison of the monomeric AMPPNP-Mg(2+)-bound GlcV structure with that of the dimeric ATP-Na(+)-bound LolD-E171Q mutant reveals a +/-20 degrees rigid body re-orientation of the ABCalpha subdomain relative to the ABCalpha/beta subdomain, accompanied by a local conformational difference in the Q-loop. We propose that these differences represent conformational changes that may have a role in the mechanism of energy-transduction and/or allosteric control of the ABC-ATPase activity in bacterial importers.