The structure of lombricine kinase: implications for phosphagen kinase conformational changes.

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

Elongation arrest by SecM via a cascade of ribosomal RNA rearrangements.

In E. coli, the SecM nascent polypeptide causes elongation arrest, while interacting with 23S RNA bases A2058 and A749-753 in the exit tunnel of the large ribosomal subunit. We compared atomic models fitted by real-space refinement into cryo-electron microscopy reconstructions of a pretranslocational and SecM-stalled E. coli ribosome complex. A cascade of RNA rearrangements propagates from the exit tunnel throughout the large subunit, affecting intersubunit bridges and tRNA positions, which in turn reorient small subunit RNA elements. Elongation arrest could result from the inhibition of mRNA.(tRNAs) translocation, E site tRNA egress, and perhaps translation factor activation at the GTPase-associated center. Our study suggests that the specific secondary and tertiary arrangement of ribosomal RNA provides the basis for internal signal transduction within the ribosome. Thus, the ribosome may itself have the ability to regulate its progression through translation by modulating its structure and consequently its receptivity to activation by cofactors.

Bias in cross-validated free R factors: mitigation of the effects of non-crystallographic symmetry.

Current methods of free R factor cross-validation assume that the structure factors of the test and working sets are independent of one another. This assumption is only an approximation when the modeled structure occupies anything less than the full asymmetric unit. Through progressive elimination of reflections from the working set, starting with those expected to be most correlated to the test set, small biases in free R can be measured, presumably because of over-sampling of the Fourier transform owing to bulk solvent in the crystal. This level of bias may be of little practical importance, but it rises to significant levels with increasing non-crystallographic symmetry owing to wider correlations between structure factors than hitherto appreciated. In the presence of 15-fold non-crystallographic symmetry, with resolutions commonly attainable in macromolecular crystallography, it may not be possible to calculate an unbiased free R factor. Methods are developed for the calculation of reduced-bias free R factors through elimination of the strongest correlations between test and working sets. With 180-fold non-crystallographic symmetry they may not be an accurate indicator of absolute quality, but they do yield the correct optimal weighting for stereochemical restraints.

Fitting of high-resolution structures into electron microscopy reconstruction images.

Dynamic macromolecular assemblies, such as ribosomes, viruses, and muscle protein complexes, are often more amenable to visualization by electron microscopy than by high-resolution X-ray crystallography or NMR. When high-resolution structures of component structures are available, it is possible to build an atomic model that gives information about the molecular interactions at greater detail than the experimental resolution, due to constraints of modeling placed upon the interpretation. There are now several competing computational methods to search systematically for orientations and positions of components that match the experimental image density, and continuing developments will be reviewed. Attention is now also moving toward the related task of optimization, with flexible and/or multifragment models and sometimes with stereochemically restrained refinement methods. This paper will review the various approaches and describe advances in the authors' methods and applications of real-space refinement.

Atomic refinement with correlated solid-state NMR restraints.

The orientation data provided by solid-state NMR can provide a great deal of structural information about membrane proteins. The quality of the information provided is, however, somewhat degraded by sign degeneracies in measurements of the dipolar coupling tensor. This is reflected in the dipolar coupling penalty function used in atomic refinement, which is less capable of properly restraining atoms when dipolar sign degeneracies are present. In this report we generate simulated solid-state NMR data using a variety of procedures, including back-calculation from crystal structures of alpha-helical and beta-sheet membrane proteins. We demonstrate that a large fraction of the dipolar sign degeneracies are resolved if anisotropic dipolar coupling measurements are correlated with anisotropic chemical shift measurements, and that all sign degeneracies can be resolved if three data types are correlated. The advantages of correlating data are demonstrated with atomic refinement of two test membrane proteins. When refinement is performed using correlated dipolar couplings and chemical shifts, perturbed structures converge to conformations with a larger fraction of correct dipolar signs than when data are uncorrelated. In addition, the final structures are closer to the original unperturbed structures when correlated data are used in the refinement. Thus, refinement with correlated data leads to improved atomic structures. The software used to correlate dipolar coupling and chemical shift data and to set up energy functions and their derivatives for refinement, CNS-SS02, is available at our web site.

Refinement of the arginine kinase transition-state analogue complex at 1.2 A resolution: mechanistic insights.

The three-dimensional crystal structure of an arginine kinase transition-state analogue complex has been refined at 1.2 A resolution, with an overall R factor of 12.3%. The current model provides a unique opportunity to analyze the structure of a bimolecular (phosphagen kinase) enzyme in its transition state. This atomic resolution structure confirms in-line transfer of the phosphoryl group and the catalytic importance of the precise alignment of the substrates. The structure is consistent with a concerted proton transfer that has been proposed for an unrelated kinase. Refinement of anisotropic temperature factors and translation-libration-screw (TLS) analyses led to the identification of four rigid groups and their prevalent modes of motion in the transition state. The relative magnitudes of the mobility of rigid groups are consistent with their proposed roles in catalysis.

An improved hydrogen bond potential: impact on medium resolution protein structures.

A new semi-empirical force field has been developed to describe hydrogen-bonding interactions with a directional component. The hydrogen bond potential supports two alternative target angles, motivated by the observation that carbonyl hydrogen bond acceptor angles have a bimodal distribution. It has been implemented as a module for a macromolecular refinement package to be combined with other force field terms in the stereochemically restrained refinement of macromolecules. The parameters for the hydrogen bond potential were optimized to best fit crystallographic data from a number of protein structures. Refinement of medium-resolution structures with this additional restraint leads to improved structure, reducing both the free R-factor and over-fitting. However, the improvement is seen only when stringent hydrogen bond selection criteria are used. These findings highlight common misconceptions about hydrogen bonding in proteins, and provide explanations for why the explicit hydrogen bonding terms of some popular force field sets are often best switched off.