Concerted release of substrate domains from GroEL by ATP is demonstrated with FRET

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.     

Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations

Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P_i, ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the γ-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.     

Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition

Although it has been known for many years that antibodies display properties characteristic of allosteric effectors, the molecular mechanisms responsible for these effects remain poorly understood. Here, we describe a single-domain antibody fragment (nanobody) that modulates protein function by constraining conformational change in the enzyme dihydrofolate reductase (DHFR). Nanobody 216 (Nb216) behaves as a potent allosteric inhibitor of DHFR, giving rise to mixed hyperbolic inhibition kinetics. The crystal structure of Nb216 in complex with DHFR reveals that the nanobody binds adjacent to the active site. Half of the epitope consists of residues from the flexible Met20 loop. This loop, which ordinarily oscillates between occluded and closed conformations during catalysis, assumes the occluded conformation in the Nb216-bound state. Using stopped flow, we show that Nb216 inhibits DHFR by stabilising the occluded Met20 loop conformation. Surprisingly, kinetic data indicate that the Met20 loop retains sufficient conformational flexibility in the Nb216-bound state to allow slow substrate turnover to occur.     

The Ever Changing Moods of Calmodulin: How Structural Plasticity Entails Transductional Adaptability

The exceptional versatility of calmodulin (CaM) three-dimensional arrangement is reflected in the growing number of structural models of CaM/protein complexes currently available in the Protein Data Bank (PDB) database, revealing a great diversity of conformations, domain organization, and structural responses to Ca^2 +. Understanding CaM binding is complicated by the diversity of target proteins sequences. Data mining of the structures shows that one face of each of the eight CaM helices can contribute to binding, with little overall difference between the Ca^2 + loaded N- and C-lobes and a clear prevalence of the C-lobe low Ca^2 + conditions. The structures reveal a remarkable variety of configurations where CaM binds its targets in a preferred orientation that can be reversed and where CaM rotates upon Ca^2 + binding, suggesting a highly dynamic metastable relation between CaM and its targets. Recent advances in structure–function studies and the discovery of CaM mutations being responsible for human diseases, besides expanding the role of CaM in human pathophysiology, are opening new exciting avenues for the understanding of the how CaM decodes Ca^2 +-dependent and Ca^2 +-independent signals.     

Energetics-based protein profiling on a proteomic scale: identification of proteins resistant to proteolysis

Native states of proteins are flexible, populating more than just the unique native conformation. The energetics and dynamics resulting from this conformational ensemble are inherently linked to protein function and regulation. Proteolytic susceptibility is one feature determined by this conformational energy landscape. As an attempt to investigate energetics of proteins on a proteomic scale, we challenged the Escherichia coli proteome with extensive proteolysis and determined which proteins, if any, have optimized their energy landscape for resistance to proteolysis. To our surprise, multiple soluble proteins survived the challenge. Maltose binding protein, a survivor from thermolysin digestion, was characterized by in vitro biophysical studies to identify the physical origin of proteolytic resistance. This experimental characterization shows that kinetic stability is responsible for the unusual resistance in maltose binding protein. The biochemical functions of the identified survivors suggest that many of these proteins may have evolved extreme proteolytic resistance because of their critical roles under stressed conditions. Our results suggest that under functional selection proteins can evolve extreme proteolysis resistance by modulating their conformational energy landscapes without the need to invent new folds, and that proteins can be profiled on a proteomic scale according to their energetic properties by using proteolysis as a structural probe.     

Analysis of Four-Way Junctions in RNA Structures

RNA secondary structures can be divided into helical regions composed of canonical Watson-Crick and related base pairs, as well as single-stranded regions such as hairpin loops, internal loops, and junctions. These elements function as building blocks in the design of diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional (3D) RNAs, we analyze existing RNA four-way junctions in terms of base-pair interactions and 3D configurations. Specifically, we identify nine broad junction families according to coaxial stacking patterns and helical configurations. We find that helices within junctions tend to arrange in roughly parallel and perpendicular patterns and stabilize their conformations using common tertiary motifs such as coaxial stacking, loop-helix interaction, and helix packing interaction. Our analysis also reveals a number of highly conserved base-pair interaction patterns and novel tertiary motifs such as A-minor-coaxial stacking combinations and sarcin/ricin motif variants. Such analyses of RNA building blocks can ultimately help in the difficult task of RNA 3D structure prediction.     

The recognition specificity of the CHD1 chromodomain with modified histone H3 peptides

Histone tail peptides comprise the flexible portion of chromatin, the substance which serves as the packaging for the eukaryotic genome. According to the histone code hypothesis, reader protein domains (chromodomains) can recognize modifications of amino acid residues within these peptides, regulating the expression of genes. We have performed simulations on models of chromodomain helicase DNA-binding protein 1 complexed with a variety of histone H3 modifications. Binding free energies for both the overall complexes and the individual residues within the protein and peptides were computed with molecular mechanics-generalized Born surface area. The simulation results agree well with experimental data and identify several chromodomain helicase DNA-binding protein 1 residues that play key roles in the interaction with each of the H3 modifications. We identified one class of protein residues that bind to H3 in all of the complexes (generally interacting hydrophobically), and a second class of residues that bind only to particular H3 modifications (generally interacting electrostatically). Additionally, we found that modifications of H3R2 and H3T3 have a dominant effect on the binding affinity; methylation of H3K4 has little effect on the interaction strength when H3R2 or H3T3 is modified. Our findings with regard to the specificity shown by the latter class of protein residues in their binding affinity to certain modifications of H3 support the histone code hypothesis.     

Hierarchical Description and Extensive Classification of Protein Structural Changes by Motion Tree

The structures of the same protein, determined under different conditions, provide clues toward understanding the role of structural changes in the protein's function. Structural changes are usually identified as rigid-body motions, which are defined using a particular threshold of rigidity, such as domain motions. However, each protein actually undergoes motions with various size and magnitude ranges. In this study, to describe protein structural changes more comprehensively, we propose a method based on hierarchical clustering. This method enables the illustration of a wide range of protein motions in a single tree diagram, named the “Motion Tree”. We applied the method to 432 proteins exhibiting large structural changes and classified their Motion Trees in terms of the characteristic indices of the trees. This classification of the Motion Trees revealed clear relationships to their protein functions. Especially, complex structural changes are significantly correlated with multi-step protein functions.     

Determinants of homodimerization specificity in histidine kinases

Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and RstB, its closest paralog in Escherichia coli. Direct binding studies showed that the cytoplasmic domains of these proteins each form specific homodimers in vitro. Using a series of chimeric proteins, we identified specificity determinants at the base of the four-helix bundle in the dimerization and histidine phosphotransfer domain. Guided by molecular coevolution predictions and EnvZ structural information, we identified sets of residues in this region that are sufficient to establish homospecificity. Mutating these residues in EnvZ to the corresponding residues in RstB produced a functional kinase that preferentially homodimerized over interacting with EnvZ. EnvZ and RstB likely diverged following gene duplication to yield two homodimers that cannot heterodimerize, and the mutants we identified represent possible evolutionary intermediates in this process.     

The interaction of cofilin with the actin filament

Cofilin is a key actin-binding protein that is critical for controlling the assembly of actin within the cell. Here, we present the results of molecular docking and dynamics studies using a muscle actin filament and human cofilin I. Guided by extensive mutagenesis results and other biophysical and structural studies, we arrive at a model for cofilin bound to the actin filament. This predicted structure agrees very well with electron microscopy results for cofilin-decorated filaments, provides molecular insight into how the known F- and G-actin sites on cofilin interact with the filament, and also suggests new interaction sites that may play a role in cofilin binding. The resulting atomic-scale model also helps us understand the molecular function and regulation of cofilin and provides testable data for future experimental and simulation work.     

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.     

Transition Path Times Measured by Single-Molecule Spectroscopy

The transition path is a tiny fraction of a molecular trajectory during which the free-energy barrier is crossed. It is a single-molecule property and contains all mechanistic information of folding processes of biomolecules such as proteins and nucleic acids. However, the transition path has been difficult to probe because it is short and rarely visited when transitions actually occur. Recent technical advances in single-molecule spectroscopy have made it possible to directly probe transition paths, which has opened up new theoretical and experimental approaches to investigating folding mechanisms. This article reviews recent single-molecule fluorescence and force spectroscopic measurements of transition path times and their connection to both theory and simulations.     

Rotations of the 2B sub-domain of E. coli UvrD helicase/translocase coupled to nucleotide and DNA binding

Escherichia coli UvrD is a superfamily 1 DNA helicase and single-stranded DNA (ssDNA) translocase that functions in DNA repair and plasmid replication and as an anti-recombinase by removing RecA protein from ssDNA. UvrD couples ATP binding and hydrolysis to unwind double-stranded DNA and translocate along ssDNA with 3′-to-5′ directionality. Although a UvrD monomer is able to translocate along ssDNA rapidly and processively, DNA helicase activity in vitro requires a minimum of a UvrD dimer. Previous crystal structures of UvrD bound to a ssDNA/duplex DNA junction show that its 2B sub-domain exists in a “closed” state and interacts with the duplex DNA. Here, we report a crystal structure of an apo form of UvrD in which the 2B sub-domain is in an “open” state that differs by an ∼ 160° rotation of the 2B sub-domain. To study the rotational conformational states of the 2B sub-domain in various ligation states, we constructed a series of double-cysteine UvrD mutants and labeled them with fluorophores such that rotation of the 2B sub-domain results in changes in fluorescence resonance energy transfer. These studies show that the open and closed forms can interconvert in solution, with low salt favoring the closed conformation and high salt favoring the open conformation in the absence of DNA. Binding of UvrD to DNA and ATP binding and hydrolysis also affect the rotational conformational state of the 2B sub-domain, suggesting that 2B sub-domain rotation is coupled to the function of this nucleic acid motor enzyme.     

The Role of L1 Stalk-tRNA Interaction in the Ribosome Elongation Cycle

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk-tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA^fMet interacts less strongly with the L1 stalk compared to elongator tRNA^Phe, as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.     

Ion Selectivity of the KcsA Channel: A Perspective from Multi-Ion Free Energy Landscapes

Potassium (K⁺) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K⁺ through cell membranes. Comprising five specific cation binding sites (S₀-S₄) formed by the backbone carbonyl groups of conserved residues common to all K⁺ channels, the narrow selectivity filter allows fast conduction of K⁺ while being highly selective for K⁺ over Na⁺. To extend our knowledge of the microscopic mechanism underlying selectivity in K⁺ channels, we characterize the free energy landscapes governing the entry and translocation of a Na⁺ or a K⁺ from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K⁺ in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K⁺ or Na⁺ in binding site S₀ or S₁ are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na⁺ than for K⁺, implying that the kinetics of ion conduction is slower when a Na⁺ enters the pore. Third, the region corresponding to binding site S₂ near the center of the narrow pore emerges as the most selective for K⁺ over Na⁺. In particular, while there is a stable minimum for K⁺ in site S₂, Na⁺ faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S₂ in the selectivity filter of K⁺ channels.     

Efficiency of Resonance Energy Transfer in Homo-Oligomeric Complexes of Proteins

A theoretical model is proposed for the apparent efficiency of fluorescence (Förster) resonance energy transfer (FRET) in mixtures of free monomers and homo-oligomeric protein complexes of uniform size. The model takes into account possible pathways for transfer of optical excitations from single donors to multiple acceptors and from multiple donors (non-simultaneously) to single acceptors. This necessary departure from the standard theory has been suggested in the literature, but it has only been successfully implemented for a few particular cases, such as for particular geometries of the oligomers. The predictions of the present theoretical model differ significantly from those of the standard theory, with the exception of the case of dimers, for which agreement is observed. This model therefore provides new insights into the FRET behavior of oligomers comprising more than two monomers, and also suggests means for determining the size of oligomeric protein complexes as well as the proportion of associated and unassociated monomers.     

Dynamics of apelin receptor/G protein coupling in living cells

During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of Gαi₂ or Gαi₃ through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi₁ subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demonstrated that stimulation of APJ with apelin-13 causes activation of Gαi₂, Gαi₃, Gαo, Gαq; among which Gαi₂, Gαi₃ were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.     

Structure of a NADPH-dependent blue fluorescent protein revealed the unique role of Gly176 on the fluorescence enhancement

A NADPH-dependent blue fluorescent protein from Vibrio vulnificus CKM-1 (BFPvv) emits blue fluorescence under UV-exposure. Previously, the BFPvvD7 mutant generated by directed evolution displayed a fourfold enhancement in fluorescent intensity. Herein, a further increase in fluorescence in the new BFPvvD8 mutant, with three additional mutations from BFPvvD7, was made. To understand the underlying mechanism of the increased fluorescent intensity of BFPvv, we solved the BFPvvD8-NADPH complex structure. Accompanied with lifetime detection, we proposed that the enhanced intensity is related to the conformational change caused by a glycine residue (Gly176) mutated to other non-glycine residues at a turn close to the NADPH binding site. We also observed the F?rster resonance energy transfer (FRET) from our BFPvvD8 to each of the GFP-like fluorescent proteins, mTFP1 and EGFP, joined by an eight-residue linker between the N-terminal of BFPvvD8 and the C-terminal of GFPs. Taken together, with the newly solved BFPvvD8 structure, our results not only provide new considerations within the rational-based protein engineering of this NADPH-dependent BFP, but also suggest that BFPvvD8 could be a potential candidate in FRET-based biosensor techniques.     

Minimalist representations and the importance of nearest neighbor effects in protein folding simulations

In order to investigate the level of representation required to simulate folding and predict structure, we test the ability of a variety of reduced representations to identify native states in decoy libraries and to recover the native structure given the advanced knowledge of the very broad native Ramachandran basin assignments. Simplifications include the removal of the entire side-chain or the retention of only the Cbeta atoms. Scoring functions are derived from an all-atom statistical potential that distinguishes between atoms and different residue types. Structures are obtained by minimizing the scoring function with a computationally rapid simulated annealing algorithm. Results are compared for simulations in which backbone conformations are sampled from a Protein Data Bank-based backbone rotamer library generated by either ignoring or including a dependence on the identity and conformation of the neighboring residues. Only when the Cbeta atoms and nearest neighbor effects are included do the lowest energy structures generally fall within 4 A of the native backbone root-mean square deviation (RMSD), despite the initial configuration being highly expanded with an average RMSD > or = 10 A. The side-chains are reinserted into the Cbeta models with minimal steric clash. Therefore, the detailed, all-atom information lost in descending to a Cbeta-level representation is recaptured to a large measure using backbone dihedral angle sampling that includes nearest neighbor effects and an appropriate scoring function.