Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal P-stalk by high-speed AFM.

In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0•(aP1•aP1)3 (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.

Large domain movements through the lipid bilayer mediate substrate release and inhibition of glutamate transporters.

Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homolog, sodium, and aspartate symporter GltPh. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of GltPh reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.

Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog.

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

Efficient Generation of Hydrazides in Proteins by RadA Split Intein.

Protein C-terminal hydrazides are useful for bioconjugation and construction of proteins from multiple fragments through native chemical ligation. To generate C-terminal hydrazides in proteins, an efficient intein-based preparation method has been developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method has been expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. It is expected that this versatile preparation method will expand the utilization of protein C-terminal hydrazides in protein preparation and modification.

Large-scale state-dependent membrane remodeling by a transporter protein.

That channels and transporters can influence the membrane morphology is increasingly recognized. Less appreciated is that the extent and free-energy cost of these deformations likely varies among different functional states of a protein, and thus, that they might contribute significantly to defining its mechanism. We consider the trimeric Na+-aspartate symporter GltPh, a homolog of an important class of neurotransmitter transporters, whose mechanism entails one of the most drastic structural changes known. Molecular simulations indicate that when the protomers become inward-facing, they cause deep, long-ranged, and yet mutually-independent membrane deformations. Using a novel simulation methodology, we estimate that the free-energy cost of this membrane perturbation is in the order of 6-7 kcal/mol per protomer. Compensating free-energy contributions within the protein or its environment must thus stabilize this inward-facing conformation for the transporter to function. We discuss these striking results in the context of existing experimental observations for this and other transporters.

Evaluating the strengths of salt bridges in the CutA1 protein using molecular dynamic simulations: a comparison of different force fields.

Ion-ion interactions (salt bridges) between favorable pairs of charged residues are important for the conformational stability of proteins. Molecular dynamic (MD) simulations are useful for elucidating the interactions among charged residues fluctuating in solution. However, the quality of MD results depends strongly on the force fields used. In this study, we compared the strengths of salt bridges among force fields by performing MD simulations using the CutA1 protein (trimer) from the hyperthermophile Pyrococcus horikoshii (PhCutA1), which has an unusually large proportion of charged residues. The force fields Chemistry at HARvard Macromolecular Mechanics (Charmm)27, Assisted Model Building and Energy Refinement (Amber)99sb, Amber14sb, GROningen Molecular Simulation (Gromos)43a1, and Gromos53a6 were used in combination with two different water models, tip3p (for Charmm27, Amber99sb, and Amber14sb) and simple point charge/extended (for Amber99sb, Gromos43a1, and Gromos53a6), yielding a total of six combinations. The RMSDs of all Cα atoms of PhCutA1 were similar among force fields, except for Charmm27, during 400-ns MD simulations at 300 K; however, the radius of gyration (Rg ) was greater for Amber99sb and shorter for Gromos43a1. The average strengths of salt bridges for each positively charged residue did not differ greatly among force fields, but the strengths at specific sites within the structure depended sensitively on the force field used. In the case of the Gromos group, positively charged residues could engage in favorable interactions with many more charged residues than in the other force fields, especially in loop regions; consequently, the apparent strength at each site was lower.

Structure of the archaeal chemotaxis protein CheY in a domain-swapped dimeric conformation.

Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection.

Distinct molecular assembly of homologous peroxiredoxins from Pyrococcus horikoshii and Thermococcus kodakaraensis.

Peroxiredoxins from Pyrococcus horikoshii (PhPrx) and Thermococcus kodakaraensis (TkPrx) are highly homologous proteins sharing 196 of the 216 residues. We previously reported a pentagonal ring-type decameric structure of PhPrx. Here, we present the crystal structure of TkPrx. Despite their homology, unlike PhPrx, the quaternary structure of TkPrx was found to be a dodecamer comprised of six homodimers arranged in a hexagonal ring-type assembly. The possibility of the redox-dependent conversion of the molecular assembly, which had been observed in PhPrx, was excluded for TkPrx based on the crystal structure of a mutant in which all of the cysteine residues were substituted with serine. The monomer structures of the dodecameric TkPrx and decameric PhPrx coincided well, but there was a slight difference in the relative orientation of the two domains. Molecular assembly of PhPrx and TkPrx in solution evaluated by gel-filtration chromatography was consistent with the crystallographic results. For both PhPrx and TkPrx, the gel-filtration elution volume slightly increased with a decrease in the protein concentration, suggesting the existence of an equilibrium state between the decameric/dodecameric ring and lower-order assembly. This structural assembly difference between highly homologous Prxs suggests a significant influence of quaternary structure on function, worthy of further exploration.

Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle.

Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5'-triphosphate (ATP)-dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions. We introduce an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, we provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins and reveal how nucleotide binding, hydrolysis, and release control switching between closed and open states. While the open conformation stabilizes the unfolded state of client proteins, the internalization of the client protein inside the chaperonin cavity speeds up its functional cycle. This approach opens new perspectives to study structures and mechanisms of various ATP-driven biological machineries in the heat of action.

Kinetic mechanism of coupled binding in sodium-aspartate symporter GltPh.

Many secondary active membrane transporters pump substrates against concentration gradients by coupling their uptake to symport of sodium ions. Symport requires the substrate and ions to be always transported together. Cooperative binding of the solutes is a key mechanism contributing to coupled transport in the sodium and aspartate symporter from Pyrococcus horikoshii GltPh. Here, we describe the kinetic mechanism of coupled binding for GltPh in the inward facing state. The first of the three coupled sodium ions, binds weakly and slowly, enabling the protein to accept the rest of the ions and the substrate. The last ion binds tightly, but is in rapid equilibrium with solution. Its release is required for the complex disassembly. Thus, the first ion serves to 'open the door' for the substrate, the last ion 'locks the door' once the substrate is in, and one ion contributes to both events.

Alteration of molecular assembly of peroxiredoxins from hyperthermophilic archaea.

Peroxiredoxin from Pyrococcus horikoshii (PhPrx) is a decameric protein formed by ring-type assembly of five dimers. To engineer the quaternary structure of PhPrx, we created a mutant PhPrx (PhPrx6m) by introducing six point mutations designed to dissociate PhPrx into dimers. Although PhPrx6m was a dimer in solution, the six dimers assembled into a dodecamer following crystallization. In the crystal structure, PhPrx6m was overoxidized, and the peroxidatic cysteine was in sulfonic acid form and two cysteines in the C-terminal region were linked by an intramolecular disulfide bond. Thus, we characterized the wild-type PhPrx overoxidized by hydrogen peroxide (PhPrxPer). Analytical ultracentrifugation showed that PhPrxPer had a higher molecular mass in solution than PhPrx. This was confirmed by analysis of the crystal structure of PhPrxPer, which was found to form a ring-type dodecamer composed of six dimers. The monomeric structures of PhPrx6m and PhPrxPer differed from that of PhPrx in the relative orientation of two domains, reflecting the number of dimers in the ring-type assembly. Unlike PhPrx, homologous peroxiredoxin from Aeropyrum pernix (ApPrx) did not undergo hexameric association. This property can be explained by the stronger connection between the two domains in ApPrx due to its C-terminal extension relative to PhPrx.

Designing better diffracting crystals of biotin carboxyl carrier protein from Pyrococcus horikoshii by a mutation based on the crystal-packing propensity of amino acids.

An alternative rational approach to improve protein crystals by using single-site mutation of surface residues is proposed based on the results of a statistical analysis using a compiled data set of 918 independent crystal structures, thereby reflecting not only the entropic effect but also other effects upon protein crystallization. This analysis reveals a clear difference in the crystal-packing propensity of amino acids depending on the secondary-structural class. To verify this result, a systematic crystallization experiment was performed with the biotin carboxyl carrier protein from Pyrococcus horikoshii OT3 (PhBCCP). Six single-site mutations were examined: Ala138 on the surface of a ß-sheet was mutated to Ile, Tyr, Arg, Gln, Val and Lys. In agreement with prediction, it was observed that the two mutants (A138I and A138Y) harbouring the residues with the highest crystal-packing propensities for ß-sheet at position 138 provided better crystallization scores relative to those of other constructs, including the wild type, and that the crystal-packing propensity for ß-sheet provided the best correlation with the ratio of obtaining crystals. Two new crystal forms of these mutants were obtained that diffracted to high resolution, generating novel packing interfaces with the mutated residues (Ile/Tyr). The mutations introduced did not affect the overall structures, indicating that a ß-sheet can accommodate a successful mutation if it is carefully selected so as to avoid intramolecular steric hindrance. A significant negative correlation between the ratio of obtaining amorphous precipitate and the crystal-packing propensity was also found.

A three-dimensional model of RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is an endoribonuclease involved in maturation of the 5'-end of tRNA. We found previously that RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNase P RNA (PhopRNA) and five protein cofactors designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38. The crystal structures of the five proteins have been determined, a three-dimensional (3-D) model of PhopRNA has been constructed, and biochemical data, including protein-RNA interaction sites, have become available. Here, this information was combined to orient the crystallographic structures of the proteins relative to their RNA binding sites in the PhopRNA model. Some alterations were made to the PhopRNA model to improve the fit. In the resulting structure, a heterotetramer composed of PhoPop5 and PhoRpp30 bridges helices P3 and P16 in the PhopRNA C-domain, thereby probably stabilizing a double-stranded RNA structure (helix P4) containing catalytic Mg2+ ions, while a heterodimer of PhoRpp21 and PhoRpp29 locates on a single-stranded loop connecting helices P11 and P12 in the specificity domain (S-domain) in PhopRNA, probably forming an appropriate conformation of the precursor tRNA (pre-tRNA) binding site. The fifth protein PhoRpp38 binds each kink-turn (K-turn) motif in helices P12.1, P12.2, and P16 in PhopRNA. Comparison of the structure of the resulting 3-D model with that of bacterial RNase P suggests transition from RNA-RNA interactions in bacterial RNase P to protein-RNA interactions in archaeal RNase P. The proposed 3-D model of P. horikoshii RNase P will serve as a framework for further structural and functional studies on archaeal, as well as eukaryotic, RNase Ps.

Structure determination of contaminant proteins using the MarathonMR procedure.

In the recent decades, essential steps of protein structure determination such as phasing by multiple isomorphous replacement and multi wave length anomalous dispersion, molecular replacement, refinement of the structure determined and its validation have been fully automated. Several computer program suites that execute all these steps as a pipeline operation have been made available. In spite of these great advances, determination of a protein structure may turn out to be a challenging task for a variety of reasons. It might be difficult to obtain multiple isomorphous replacement or multi wave length anomalous dispersion data or the crystal may have defects such as twinning or pseudo translation. Apart from these usual difficulties, more frequent difficulties have been encountered in recent years because of the large number of projects handled by structural biologists. These new difficulties usually result from contamination of the protein of interest by other proteins or presence of proteins from pathogenic organisms that could withstand the antibiotics used to prevent bacterial contamination. It could also be a result of poor book keeping. Recently, we have developed a procedure called MarathonMR that has the power to resolve some of these problems automatically. In this communication, we describe how the MarathonMR was used to determine four different protein structures that had remained elusive for several years. We describe the plausible reasons for the difficulties encountered in determining these structures and point out that the method presented here could be a validation tool for protein structures deposited in the protein data bank.

Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold.

Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 µM to 1.6 µM, according to surface plasmon resonance data. While L4 has 1-2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1-0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.

The Structure of an Archaeal ß-Glucosaminidase Provides Insight into Glycoside Hydrolase Evolution.

The archaeal exo-ß-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal structure of GlmA in complex with glucosamine at 1.27 Å resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 ß-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 ß-galactosidases. We found that upon dimerization, the active site of GlmA changes shape, enhancing its ability to hydrolyze the smaller substrate in a manner similar to that of homotrimeric GH42 ß-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the "evolutionary heritage residue" it shares with GH35 ß-galactosidase. Our study suggests that GH35 and GH42 ß-galactosidases evolved by exploiting the structural features of GlmA.

New insights into the mechanism of substrates trafficking in Glyoxylate/Hydroxypyruvate reductases.

Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.

Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

GltPh from Pyrococcus horikoshii is a homotrimeric Na(+)-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na(+) and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na(+) binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m(-1)s(-1)). At least two sodium ions bind before aspartate. The binding of Na(+) requires a very high activation energy (Ea 106.8 kJ mol(-1)) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na(+) concentration present. Binding of aspartate was not observed in the absence of Na(+), whereas in the presence of high Na(+) concentrations (above the Kd for Na(+)) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 10(5) m(-1)s(-1)), with low Ea and Q10 values (42.6 kJ mol(-1) and 1.8, respectively). We conclude that Na(+) binding is most likely the rate-limiting step for substrate binding.

Transport domain unlocking sets the uptake rate of an aspartate transporter.

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signalling and preventing glutamate-mediated excitotoxicity. Crystallographic studies of a glutamate transporter homologue from the archaeon Pyrococcus horikoshii, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two mutations introduced to impart characteristics of the human transporter exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motion and substrate uptake. Crystallographic and computational investigations corroborated these findings by revealing that the 'humanizing' mutations favour structurally 'unlocked' intermediate states in the transport cycle exhibiting increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.