Paul C. Whitford

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.

An all‐atom structure‐based potential for proteins: Bridging minimal models with all‐atom empirical forcefields

Protein dynamics take place on many time and length scales. Coarse‐grained structure‐based

SMOG@ctbp: simplified deployment of structure-based models in GROMACS.

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Biomolecular dynamics: order?disorder transitions and energy landscapes

models utilize the funneled energy landscape theory of protein folding to provide an understanding of both long time and long length scale dynamics. All‐atom empirical forcefields with explicit solvent can elucidate our understanding of short time dynamics with high energetic and structural resolution. Thus, structure‐based models with atomic details included can be used to bridge our understanding between these two approaches. We report on the robustness of folding mechanisms in one such all‐atom model. Results for the B domain of Protein A, the SH3 domain of C‐Src Kinase, and Chymotrypsin Inhibitor 2 are reported. The interplay between side chain packing and backbone folding is explored. We also compare this model to a Cα structure‐based model and an all‐atom empirical forcefield. Key findings include: (1) backbone collapse is accompanied by partial side chain packing in a cooperative transition and residual side chain packing occurs gradually with decreasing temperature, (2) folding mechanisms are robust to variations of the energetic parameters, (3) protein folding free‐energy barriers can be manipulated through parametric modifications, (4) the global folding mechanisms in a Cα model and the all‐atom model agree, although differences can be attributed to energetic heterogeneity in the all‐atom model, and (5) proline residues have significant effects on folding mechanisms, independent of isomerization effects. Because this structure‐based model has atomic resolution, this work lays the foundation for future studies to probe the contributions of specific energetic factors on protein folding and function. Proteins 2009.

Extracting function from a β-trefoil folding motif

Molecular dynamics simulations with coarse-grained and/or simplified Hamiltonians are an effective means of capturing the functionally important long-time and large-length scale motions of proteins and RNAs. Structure-based Hamiltonians, simplified models developed from the energy landscape theory of protein folding, have become a standard tool for investigating biomolecular dynamics. SMOG@ctbp is an effort to simplify the use of structure-based models. The purpose of the web server is two fold. First, the web tool simplifies the process of implementing a well-characterized structure-based model on a state-of-the-art, open source, molecular dynamics package, GROMACS. Second, the tutorial-like format helps speed the learning curve of those unfamiliar with molecular dynamics. A web tool user is able to upload any multi-chain biomolecular system consisting of standard RNA, DNA and amino acids in PDB format and receive as output all files necessary to implement the model in GROMACS. Both Cα and all-atom versions of the model are available. SMOG@ctbp resides at http://smog.ucsd.edu.

Excited states of ribosome translocation revealed through integrative molecular modeling

While the energy landscape theory of protein folding is now a widely accepted view for understanding how relatively weak molecular interactions lead to rapid and cooperative protein folding, such a framework must be extended to describe the large-scale functional motions observed in molecular machines. In this review, we discuss (1) the development of the energy landscape theory of biomolecular folding, (2) recent advances toward establishing a consistent understanding of folding and function and (3) emerging themes in the functional motions of enzymes, biomolecular motors and other biomolecular machines. Recent theoretical, computational and experimental lines of investigation have provided a very dynamic picture of biomolecular motion. In contrast to earlier ideas, where molecular machines were thought to function similarly to macroscopic machines, with rigid components that move along a few degrees of freedom in a deterministic fashion, biomolecular complexes are only marginally stable. Since the stabilizing contribution of each atomic interaction is on the order of the thermal fluctuations in solution, the rigid body description of molecular function must be revisited. An emerging theme is that functional motions encompass order-disorder transitions and structural flexibility provides significant contributions to the free energy. In this review, we describe the biological importance of order-disorder transitions and discuss the statistical-mechanical foundation of theoretical approaches that can characterize such transitions.

Conformational Transitions in Adenylate Kinase ALLOSTERIC COMMUNICATION REDUCES MISLIGATION

Despite having remarkably similar three-dimensional structures and stabilities, IL-1β promotes signaling, whereas IL-1Ra inhibits it. Their energy landscapes are similar and have coevolved to facilitate competitive binding to the IL-1 receptor. Nevertheless, we find that IL-1Ra folds faster than IL-1β. A structural alignment of the proteins shows differences mainly in two loops, a β-bulge of IL-1β and a loop in IL-1Ra that interacts with residue K145 and connects β-strands 11 and 12. Bioassays indicate that inserting the β-bulge from IL-1β confers partial signaling capability onto a K145D mutant of IL-1Ra. Based on the alignment, mutational assays and our computational folding results, we hypothesize that functional regions are not central to the β-trefoil motif and cause slow folding. The IL-1β β-bulge facilitates activity and replacing it by the IL-1Ra β-turn results in a hybrid protein that folds faster than IL-1β. Inserting the β11–β12 connecting-loop, which aids inhibition, into either IL-1β or the hybrid protein slows folding. Thus, regions that aid function (either through activity or inhibition) can be inferred from folding traps via structural differences. Mapping functional properties onto the numerous folds determined in structural genomics efforts is an area of intense interest. Our studies provide a systematic approach to mapping the functional genomics of a fold family.

Mutations as trapdoors to two competing native conformations of the Rop-dimer

The dynamic nature of biomolecules leads to significant challenges when characterizing the structural properties associated with function. While X-ray crystallography and imaging techniques (such as cryo-electron microscopy) can reveal the structural details of stable molecular complexes, strategies must be developed to characterize configurations that exhibit only marginal stability (such as intermediates) or configurations that do not correspond to minima on the energy landscape (such as transition-state ensembles). Here, we present a methodology (MDfit) that utilizes molecular dynamics simulations to generate configurations of excited states that are consistent with available biophysical and biochemical measurements. To demonstrate the approach, we present a sequence of configurations that are suggested to be associated with transfer RNA (tRNA) movement through the ribosome (translocation). The models were constructed by combining information from X-ray crystallography, cryo-electron microscopy, and biochemical data. These models provide a structural framework for translocation that may be further investigated experimentally and theoretically to determine the precise energetic character of each configuration and the transition dynamics between them.

Energy landscape along an enzymatic reaction trajectory: hinges or cracks?

Large conformational changes in the LID and NMP domains of adenylate kinase (AKE) are known to be key to ligand binding and catalysis, yet the order of binding events and domain motion is not well understood. Combining the multiple available structures for AKE with the energy landscape theory for protein folding, a theoretical model was developed for allostery, order of binding events, and efficient catalysis. Coarse-grained models and nonlinear normal mode analysis were used to infer that intrinsic structural fluctuations dominate LID motion, whereas ligand-protein interactions and cracking (local unfolding) are more important during NMP motion. In addition, LID-NMP domain interactions are indispensable for efficient catalysis. LID domain motion precedes NMP domain motion, during both opening and closing. These findings provide a mechanistic explanation for the observed 1:1:1 correspondence between LID domain closure, NMP domain closure, and substrate turnover. This catalytic cycle has likely evolved to reduce misligation, and thus inhibition, of AKE. The separation of allosteric motion into intrinsic structural fluctuations and ligand-induced contributions can be generalized to further our understanding of allosteric transitions in other proteins.

Connecting the Kinetics and Energy Landscape of tRNA Translocation on the Ribosome

Conformational transitions play a central role in regulating protein function. Structure-based models with multiple basins have been used to understand the mechanisms governing these transitions. A model able to accommodate multiple folding basins is proposed to explore the mutational effects in the folding of the Rop-dimer (Rop). In experiments, Rop mutants show unusually strong increases in folding rates with marginal effects on stability. We investigate the possibility of two competing conformations representing a parallel (P) and the wild-type antiparallel (AP) arrangement of the monomers as possible native conformations. We observe occupation of both distinct states and characterize the transition pathways. An interesting observation from the simulations is that, for equivalent energetic bias, the transition to the P basin (non-wild-type basin) shows a lower free-energy barrier. Thus, the rapid kinetics observed in experiments appear to be the result of two competing states with different kinetic behavior, triggered upon mutation by the opening of a trapdoor arising from Rops symmetric structure. The general concept of having competing conformations for the native state goes beyond explaining Rops mutational behaviors and can be applied to other systems. A switch between competing native structures might be triggered by external factors to allow, for example, allosteric control or signaling.