Long-Range Coupling in an Allosteric Receptor Revealed by Mutant Cycle Analysis

The functional coupling of residues that are far apart in space is the quintessential property of allosteric proteins. For example, in Cys-loop receptors, the gating of an intrinsic ion channel is allosterically regulated by the binding of small molecule neurotransmitters 50-60 Å from the channel gate. Some residues near the binding site must have as their primary function the communication of the binding event to the gating region. These gating pathway residues are essential to function, but their identification and characterization can be challenging. This work introduces a simple strategy, derived from mutant cycle analysis, for identifying gating pathway residues using macroscopic measurements alone. In the exemplar Cys-loop receptor, the nicotinic acetylcholine receptor, a well-characterized reporter mutation (βL9′S) known to impact gating, was combined with mutations of target residues in the ligand-binding domain hypothesized or previously found to be functionally significant. A mutant cycle analysis of the macroscopic EC₅₀ measurements can then provide insights into the role of the target residue. This new method, elucidating long-range functional coupling in allosteric receptors, can be applied to several reporter mutations in a wide variety of receptors to identify previously characterized and novel mutations that impact the gating pathway. We support our interpretation of macroscopic data with single-channel studies. Elucidating long-range functional coupling in allosteric receptors should be broadly applicable to determining functional roles of residues in allosteric receptors.     

Allosteric Transitions of Supramolecular Systems Explored by Network Models: Application to Chaperonin GroEL

Identification of pathways involved in the structural transitions of biomolecular systems is often complicated by the transient nature of the conformations visited across energy barriers and the multiplicity of paths accessible in the multidimensional energy landscape. This task becomes even more challenging in exploring molecular systems on the order of megadaltons. Coarse-grained models that lend themselves to analytical solutions appear to be the only possible means of approaching such cases. Motivated by the utility of elastic network models for describing the collective dynamics of biomolecular systems and by the growing theoretical and experimental evidence in support of the intrinsic accessibility of functional substates, we introduce a new method, adaptive anisotropic network model (aANM), for exploring functional transitions. Application to bacterial chaperonin GroEL and comparisons with experimental data, results from action minimization algorithm, and previous simulations support the utility of aANM as a computationally efficient, yet physically plausible, tool for unraveling potential transition pathways sampled by large complexes/assemblies. An important outcome is the assessment of the critical inter-residue interactions formed/broken near the transition state(s), most of which involve conserved residues.     

A New Mode of Dimerization of Allosteric Enzymes with ACT Domains Revealed by the Crystal Structure of the Aspartate Kinase from Cyanobacteria

Aspartate kinases (AKs) can be divided in two subhomology divisions, AKα and AKβ, depending on the presence of an extra sequence of about 60 amino acids, which is found only in the N-terminus of all AKα's. To date, the structures of AKα failed to provide a role for this additional N-terminal sequence. In this study, the structure of the AKβ from the Cyanobacteria Synechocystis reveals that this supplementary sequence is linked to the dimerization mode of AKs. Its absence in AKβ leads to the dimerization by the catalytic domain instead of involving the ACT domains [Pfam 01842; small regulatory domains initially found in AK, chorismate mutase and TyrA (prephenate dehydrogenase)] as observed in AKα. Thus, the structural analysis of the Synechocystis AKβ revealed a dimer with a novel architecture. The four ACT domains of each monomer interact together and do not make any contact with those of the second monomer. The enzyme is inhibited synergistically by threonine and lysine with the binding of threonine first. The interaction between ACT1 and ACT4 or between ACT2 and ACT3 generates a threonine binding site and a lysine binding site at each interface, making a total of eight regulatory sites per dimer and allowing a fine-tuning of the AK activity by the end products, threonine and lysine.     

Molecular Mechanism of Allosteric Communication in Hsp70 Revealed by Molecular Dynamics Simulations

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.     

Computational Modeling of Allosteric Regulation in the Hsp90 Chaperones: A Statistical Ensemble Analysis of Protein Structure Networks and Allosteric Communications

A fundamental role of the Hsp90 chaperone in regulating functional activity of diverse protein clients is essential for the integrity of signaling networks. In this work we have combined biophysical simulations of the Hsp90 crystal structures with the protein structure network analysis to characterize the statistical ensemble of allosteric interaction networks and communication pathways in the Hsp90 chaperones. We have found that principal structurally stable communities could be preserved during dynamic changes in the conformational ensemble. The dominant contribution of the inter-domain rigidity to the interaction networks has emerged as a common factor responsible for the thermodynamic stability of the active chaperone form during the ATPase cycle. Structural stability analysis using force constant profiling of the inter-residue fluctuation distances has identified a network of conserved structurally rigid residues that could serve as global mediating sites of allosteric communication. Mapping of the conformational landscape with the network centrality parameters has demonstrated that stable communities and mediating residues may act concertedly with the shifts in the conformational equilibrium and could describe the majority of functionally significant chaperone residues. The network analysis has revealed a relationship between structural stability, global centrality and functional significance of hotspot residues involved in chaperone regulation. We have found that allosteric interactions in the Hsp90 chaperone may be mediated by modules of structurally stable residues that display high betweenness in the global interaction network. The results of this study have suggested that allosteric interactions in the Hsp90 chaperone may operate via a mechanism that combines rapid and efficient communication by a single optimal pathway of structurally rigid residues and more robust signal transmission using an ensemble of suboptimal multiple communication routes. This may be a universal requirement encoded in protein structures to balance the inherent tension between resilience and efficiency of the residue interaction networks.     

Allosteric Coupling between the Lid and Interdomain Linker in DnaK Revealed by Inhibitor Binding Studies

The molecular chaperone DnaK assists protein folding and refolding, translocation across membranes, and regulation of the heat shock response. In Escherichia coli, the protein is a target for insect-derived antimicrobial peptides, pyrrhocoricins. We present here the X-ray crystallographic analysis of the E. coli DnaK substrate-binding domain in complex with pyrrhocoricin-derived peptide inhibitors. The structures show that pyrrhocoricins act as site-specific, dual-mode (competitive and allosteric) inhibitors, occupying the substrate-binding tunnel and disrupting the latch between the lid and the β-sandwich. Our structural analysis revealed an allosteric coupling between the movements of the lid and the interdomain linker, identifying a previously unknown mechanism of the lid-mediated regulation of the chaperone cycle.DnaK is the bacterial molecular chaperone from the Hsp70 (70-kDa heat shock protein) family that assists many cellular processes involving proteins in their nonnative conformations, such as folding of newly synthesized proteins, protein translocation across membranes, refolding of misfolded and aggregated proteins, degradation of unstable proteins, and regulation of the heat shock response (for reviews, see references 10, 28, and 42). The chaperone function of DnaK is based on its ability to transiently bind to exposed stretches of hydrophobic residues in partially or fully unfolded proteins in an ATP-controlled fashion, thereby preventing aggregation and misfolding. DnaK recognizes short peptide sequences containing up to five consecutive hydrophobic residues (with leucine found frequently in the middle), flanked preferentially by basic residues (42, 43). The molecular-chaperone activity is functionally linked with ATP hydrolysis; the substrate-binding and release cycle is driven by the switching between the ATP-bound state, with low affinity and a high exchange rate for substrates, and the ADP-bound state, with high affinity and a low exchange rate for substrates.In vivo, DnaK activity is supported by two cochaperones, GrpE, which facilitates the ADP/ATP exchange, and DnaJ, which stimulates ATP hydrolysis and thus aids the peptide capture (10, 25, 28, 38, 54). DnaJ itself also recognizes exposed stretches of hydrophobic residues in partially unfolded or denatured proteins, with specificity overlapping with that of DnaK (44). It is therefore thought that DnaJ serves as a scanning factor for DnaK by binding specific unfolded substrates and presenting them to the ATP-bound form of DnaK.Escherichia coli DnaK is composed of two domains: an N-terminal ATPase domain (residues 1 to 387) and a C-terminal substrate-binding domain (SBD) (residues 388 to 638) (10). The latter is made up of an 18-kDa β-sandwich subdomain that holds the substrate-binding cleft and a C-terminal α-helical-bundle “lid” subdomain that stabilizes the complex with the peptide substrate and controls the accessibility of the peptide binding site but does not interact with the substrate directly (5, 55). Removal of the lid subdomain by truncation decreases the affinity of DnaK for polypeptide substrates, primarily by increasing the dissociation rates (9, 28, 46, 47).The activities of the ATPase and the SBDs are allosterically coupled. ATP binding induces a global conformational change that results in the docking of the ATPase domain onto the SBD and opening of the latter, thus triggering the release of a peptide substrate (45, 46). Peptide binding, in turn, accelerates DnaK/DnaJ-mediated ATP hydrolysis, followed by trapping of the substrate and dissociation of the ATPase domain from the SBD. Upon the subsequent GrpE-mediated exchange of ADP for ATP, DnaK returns to the beginning of its molecular-chaperone cycle (references 6 and 28 and references therein). In this manner, DnaK alternates between the “open” (low-peptide-affinity) and closed (high-peptide-affinity) states. The detailed molecular mechanism of the allosteric interdomain communication is unknown, although the previous mutagenesis studies identified the conserved interdomain linker VLLL (389 to 392) (25), the segment 507 to 537 (32), and residue K414 (31) on the SBD surface as the structural elements that are required for signal transmission.Previous X-ray crystallographic and nuclear magnetic resonance studies of the SBD complex with the heptapeptide NR (NRLLLTG) provided an insight into the structural basis for the substrate recognition and amino acid sequence specificity of DnaK (48, 55). The peptide has been shown to bind in a short tunnel formed by the loops of the β-sandwich subdomain of the SBD in an extended conformation through hydrophobic and van der Waals side chain interactions and hydrogen bonds between the peptide backbones of the substrate and the SBD. These structures rationalized the ability of DnaK to differentiate between native and nonnative protein conformers by recognizing structural features common to nascent chains: an accessible peptide backbone and solvent-exposed aliphatic side chains.DnaK has been identified as a molecular target of pyrrhocoricin (l-PYR) (VDKGSYLPRPTPPRPIYNRN), an insect-derived antibacterial peptide (13, 23, 34). Nontoxicity to mammalian cells and good serum stability of l-PYR-derived peptides make them promising drug candidates in treating emerging/re-emerging antimicrobial-resistant bacterial pathogens (15, 35) and highlight the importance of detailed structural characterization of inhibitor-protein interactions with a view to rational drug design. This paper describes the first crystal structures of the E. coli DnaK SBD, truncated at the C terminus (residues 389 to 607), in complex with two l-PYR-derived peptidic inhibitors, one of which (the short peptide) displays nanomolar affinity for nucleotide-free E. coli DnaK (K_d [dissociation constant] = 5.5 nM, the lowest that has ever been reported for any peptide [M. Liebscher, unpublished data]).     

The Dynamic Conformational Cycle of the Group I Chaperonin C-Termini Revealed via Molecular Dynamics Simulation

Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins.     

Streptococcus suis Serotypes Characterized by Analysis of Chaperonin 60 Gene Sequences

Streptococcus suis is an important pathogen of swine which occasionally infects humans as well. There are 35 serotypes known for this organism, and it would be desirable to develop rapid methods methods to identify and differentiate the strains of this species. To that effect, partial chaperonin 60 gene sequences were determined for the 35 serotype reference strains of S. suis. Analysis of a pairwise distance matrix showed that the distances ranged from 0 to 0.275 when values were calculated by the maximum-likelihood method. For five of the strains the distances from serotype 1 were greater than 0.1, and for two of these strains the distances were were more than 0.25, suggesting that they belong to a different species. Most of the nucleotide differences were silent; alignment of protein sequences showed that there were only 11 distinct sequences for the 35 strains under study. The chaperonin 60 gene phylogenetic tree was similar to the previously published tree based on 16S rRNA sequences, and it was also observed that strains with identical chaperonin 60 gene sequences tended to have identical 16S rRNA sequences. The chaperonin 60 gene sequences provided a higher level of discrimination between serotypes than the 16S RNA sequences provided and could form the basis for a diagnostic protocol.     

Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry

1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k_cat = 0.005 s⁻¹) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.     

Atomistic Detailed Mechanism and Weak Cation-Conducting Activity of HIV-1 Vpu Revealed by Free Energy Calculations

The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na⁺ and K⁺ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel.     

REACH Coarse-Grained Normal Mode Analysis of Protein Dimer Interaction Dynamics

The REACH (realistic extension algorithm via covariance Hessian) coarse-grained biomolecular simulation method is a self-consistent multiscale approach directly mapping atomistic molecular dynamics simulation results onto a residue-scale model. Here, REACH is applied to calculate the dynamics of protein-protein interactions. The intra- and intermolecular fluctuations and the intermolecular vibrational densities of states derived from atomistic molecular dynamics are well reproduced by the REACH normal modes. The phonon dispersion relations derived from the REACH lattice dynamics model of crystalline ribonuclease A are also in satisfactory agreement with the corresponding all-atom results. The REACH model demonstrates that increasing dimer interaction strength decreases the translational and rotational intermolecular vibrational amplitudes, while their vibrational frequencies are relatively unaffected. A comparative study of functionally interacting biological dimers with crystal dimers, which are formed artificially via crystallization, reveals a relation between their static structures and the interprotein dynamics: i.e., the consequence of the extensive interfaces of biological dimers is reduction of the intermonomer translational and rotational amplitudes, but not the frequencies.     

Envisioning the Loop Movements and Rotation of the Two Subdomains of Dihydrofolate Reductase by Elastic Normal Mode Analysis

Abstract

Normal mode analysis, using the elastic network model, has been employed to envision the low frequency normal mode motion trends in the structures of five intermediates and a transition state in the kinetic pathway of E. coli dihydrofolate reductase (DHFR). Five of the reaction pathway analog structures and a crystal structure resembling the transition state, using X-ray analyses determined by Kraut et al., have been adapted as structural models. The motions that poise pathways of the M20 loop transitions from closed to occluded conformations and sub domain rotation to close the substrate cleft, have been predicted and envisioned for the first time by this study. Pathway entries to the movement of the substrate binding cleft helices are also envisioned. These motions play roles in transition structure stabilization and in regulating the release of the product tetrahydrofolate (THF). The motions observed push the ground state conformation of each intermediate towards a higher energy sub state conformation. A set of conserved residues involved in the catalytic reactions and conformational changes, previously studied by kinetic, theoretical and NMR, have been analyzed. The importance of these motions in terms of protein dynamics are revealed and envisioned by the normal mode analysis. Additional residues are proposed as candidates for further study of their potential promotional function.     

Insights into Substrate Specificity of Geranylgeranyl Reductases Revealed by the Structure of Digeranylgeranylglycerophospholipid Reductase, an Essential Enzyme in the Biosynthesis of Archaeal Membrane Lipids

Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with flavin adenine dinucleotide (FAD) and a bacterial lipid. The DGGR structure can be assigned to the well-studied, p-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two, narrow, curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the enzyme and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of geranylgeranyl reductases. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.     

Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis

Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral. We report herein that high-throughput chemical experiments can isolate an RNA’s multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M²) and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states’ structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M²-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10%) and conservative error estimates. In experimental benchmarks, M²-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M²-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.     

Archaeal Community Revealed by 16s rRNA and Fluorescence in situ Hybridization in a Sulphuric Hydrothermal Hot Spring, Northern Taiwan

Summary The archaeal community composition of Yangmingshan National Park in northern Taiwan was investigated by 16S rRNA and fluorescence in situ hybridization (FISH). Optimization of tetrameric restriction enzyme (TRE) was performed to achieve efficient digestion and differentiation in the restriction fragment length polymorphism (RFLP) fragments, and AciI, BstUI and RsaI were shown to be the optimal TREs for TRE-RFLP. Nine clones were obtained in the studies, with clones M70 and M6 being found to be phylogenetically affiliated to Sulfolobus and Caldisphaera in domain Crenarchaeota, respectively, whereas seven other clones were found to be affiliated to an uncultured and unidentified archaeon isolated from thermoacidic environments. In FISH, soil and water region cells were hybridized with DAPI (4′, 6-diamidino-2-phenylindole) and specific fluorescently labelled probes. 15.69 and 7.16% of the DAPI-stained cells hybridized with universal archaeal probe ARC915 and sulphate-reducing bacterial probe SRB385, respectively.     

The Binding Mode of ATP Revealed by the Solution Structure of the N-domain of Human ATP7A

We report the solution NMR structures of the N-domain of the Menkes protein (ATP7A) in the ATP-free and ATP-bound forms. The structures consist of a twisted antiparallel six-stranded β-sheet flanked by two pairs of α-helices. A protein loop of 50 amino acids located between β3 and β4 is disordered and mobile on the subnanosecond time scale. ATP binds with an affinity constant of (1.2 ± 0.1) × 10⁴ m⁻¹ and exchanges with a rate of the order of 1 × 10³ s⁻¹. The ATP-binding cavity is considerably affected by the presence of the ligand, resulting in a more compact conformation in the ATP-bound than in the ATP-free form. This structural variation is due to the movement of the α1-α2 and β2-β3 loops, both of which are highly conserved in copper(I)-transporting P_IB-type ATPases. The present structure reveals a characteristic binding mode of ATP within the protein scaffold of the copper(I)-transporting P_IB-type ATPases with respect to the other P-type ATPases. In particular, the binding cavity contains mainly hydrophobic aliphatic residues, which are involved in van der Waal''s interactions with the adenine ring of ATP, and a Glu side chain, which forms a crucial hydrogen bond to the amino group of ATP.