Control by phosphorylation

The two examples of phospho and dephospho proteins for which structural data were previously available (glycogen phosphorylase and isocitrate dehydrogenase) demonstrated two different mechanisms for control. In glycogen phosphorylase, activation by phosphorylation results in long-range allosteric changes. In isocitrate dehydrogenase, inhibition by phosphorylation is achieved by an electrostatic blocking mechanism with no conformational changes. During the past year, the structures of the phospho and dephospho forms of two more proteins, the cell cycle protein kinase CDK2 and yeast glycogen phosphorylase, have been determined. The new results highlight the importance of the phosphoamino acids both in the organization of local regions of protein structure through phosphate—arginine interactions and in the promotion of long-range conformational responses.     

Structure, evolution and action of vitamin B6-dependent enzymes

The number of known three-dimensional structures of vitamin B₆-dependent enzymes has doubled in the past two years. A fourth type of fold for B₆-dependent enzymes, involving a TIM-barrel domain, has been discovered. Alanine racemase is the first known representative of this new fold. Significant progress has been made in understanding the allosteric effects in the tryptophan synthase reaction.     

Three-dimensional structural studies on fragments of fibrinogen and fibrin

Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant γC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from fibrin.     

Glutamine PRPP amidotransferase: snapshots of an enzyme in action

Recent studies of glutamine PRPP amidotransferase have provided a new understanding of the function and mechanism of this rather complicated enzyme that may be a paradigm for other complex enzymes. New insights have been gained into the mechanisms of catalysis in the active sites of the two half-reactions, catalytic coupling, allosteric control by feedback inhibitors and the channeling of reaction and metabolic intermediates.     

Searching for new allosteric sites in enzymes

The discovery of new allosteric sites generates opportunities for the identification of novel pharmaceuticals and increases our understanding of basic biological processes. Increasingly, allosteric sites are being discovered in various families of proteins by several methods, paving the way for the development of entirely new classes of drugs with a wide range of chemotypes. New allosteric sites in enzymes have been discovered both incidentally and by directed means, and the mechanisms by which allosteric activation and inhibition occur at these sites have been investigated. By exploring recent structurally well-characterized examples, trends begin to emerge for both the modes of binding and mechanisms of inhibition.     

The dynamics of cyclin dependent kinase structure

In the past year, several new crystal structures have provided exciting insights into the conformational changes underlying the regulation of cyclin-dependent kinases. We now understand the structural basis of many of the mechanisms by which cyclin-dependent kinases are regulated, including activation by cycling binding and phosphorylation, inhibition by the inhibitor p27, and binding by the CKS proteins.     

Antifreeze proteins

Antifreeze proteins comprise a structurally diverse class of proteins that inhibit the growth of ice. Recently, new AFP types have been discovered; more active AFPs have been isolated; antecedents have been recognized supporting the notion of recent, multiple origins; and detailed structures have emerged leading to models for their adsorption to ice     

Structures of Src-family tyrosine kinases

The crystal structures of three Src-family tyrosine kinases have been determined recently. The structure of the catalytic domain of Lck has been determined in the active autophosphorylated state. The structures of larger constructs of c-Src and Hck, containing the SH3, SH2 and catalytic domains, as well as a C-terminal regulatory tail, have been determined in the down-regulated state, phosphorylated in the C-terminal tail. A comparison of these structures leads to an unanticipated mechanism for the regulation of catalytic activity by cooperative interactions between the SH2, SH3 and catalytic domains.     

Calcineurin—immunosuppressor complexes

Crystal structures of the Ser/Thr phosphatase calcineurin (protein phosphatase 2B) have recently been solved by X-ray crystallography, both in the free-protein state, and complexed with the immunophilin/immunosuppressant FKBP12/FK506. Core elements of the calcineurin phosphatase have been found to be similar to the corresponding elements of Ser/Thr phosphatase 1 and purple acid phosphatase. The structures provide a basis for understanding calcineurin inhibition by a ternary complex of immunophilin and immunosuppressant proteins.     

Protein-biotin interactions

The three-dimensional structures of several biotin-binding proteins are now known, giving insights into the molecular architecture of the binding sites for biotin. In combination with biochemical and computational approaches, these structural insights provide the basis for our present understanding of biotin—protein interactions which, in some cases, give rise to spectacular binding constants.     

Mechanisms of DNA bending

Genome packaging and gene regulation require DNA bending. Recent developments in the elucidation of the mechanisms involved in DNA bending include new X-ray structures (most notably that of the mammalian nucleosome) wherein DNA is bent, controversy surrounding interpretation of DNA-bending experiments with basic-leucine zipper proteins, studies of electrostatic effects in DNA bending, and the design of artificial DNA-bending ligands.     

The diverse world of coenzyme A binding proteins

Coenzyme A is involved in a number of important metabolic pathways. Recently the structures of several coenzyme A binding proteins have been determined. We compare in some detail the structures of seven different coenzyme A protein complexes. These seven proteins all have distinctly different folds.     

Messages from ultrahigh resolution crystal structures

The ever growing availability of macromolecular crystal structures determined at atomic resolution has now reached a critical size, making it possible to obtain statistically unbiased data on both protein stereochemistry and the validity of the parameters used in their refinement. Besides the determination of the precise geometry of proteins and their active sites, high resolution structures have made it possible to check the application of normal mode calculations, to calculate charge density distributions and to analyze hydration shells around protein molecules. Even if only a few structures involve protein complexes, either with ligands or prosthetic groups, the information obtained in these cases is of great interest for obtaining the physical parameters of these interactions.     

RNA catalysis

Our understanding of the relationship between the structure of RNA and its catalytic activity has advanced significantly in the past year. These advances include time-resolved crystallographic studies on the hammerhead ribozyme, as well as new structures of a group I intron, a lead(II)-cleavage ribozyme, a hepatitis delta virus ribozyme, and components of the spliceosome machinery and the peptidyl transferase center of the ribosome and, most significantly, the structure of the ribosome itself.     

The representation of temporal information in perception and motor control

The representation of temporal information can be examined from both a neurological and a computational perspective. Recent evidence suggests that two subcortical structures, the cerebellum and basal ganglia, play a critical role in the timing of both movement and perception. At a computational level, models of an internal clock have been developed in which timing is based on either endogeneous oscillatory processes or distributed interval-based representations derived from relatively slow physiological processes.     

T-cell receptor structure and TCR complexes

The first crystal structures of intact T-cell receptors (TCRs) and their complexes with MHC peptide antigens (pMHC) were reported during the past year, along with those of a single-chain TCR Fv fragment and a β-chain complexed with two different bacterial superantigens. These structures have shown the similarities and differences in the architecture of the antigen-binding regions of TCRs and antibodies, and how the TCR interacts with pMHC ligands as well as with superantigens     

Will combinatorial chemistry deliver real medicines?

Over the next decade, the impact of library synthesis will play a major role in shortening the lead optimization phase of drug discovery. The prognosis for combinatorial chemistry to discover fundamentally different new classes of therapeutically active small molecules against some of the more difficult biological targets is less certain. Expectations are high because the technology potentially allows us to sample available drug space by synthesizing all possible small molecule ligands (variously estimated to be between 10³⁰–10⁵⁰ compounds). Some caution is advised, however, since, despite recent increases in high-throughput screening of substantially greater numbers of synthetic compounds and natural products, we are not routinely finding a plethora of new structures. The outcome may be that combinatorial chemistry offers us the ability to work faster on finding ligands for well-established tractable targets, such as G-protein-coupled receptors, ion channels or proteases, rather than, say, the more complex protein—protein interactions which from the majority of targets in signal transduction pathways.     

The structural basis of negative cooperativity: receptors and enzymes

Crystal structures of the negatively cooperative aspartate receptor caught at intermediate stages in the binding process help to elucidate structural factors involved in ligand binding. The frequency of occurrence of negatively cooperative proteins suggests that sequential changes in binding patterns will be extensive in positively cooperative as well as in negatively cooperative and no cooperativity proteins.     

Implications of sequencing bacterial genomes for pathogenesis and vaccine development

Improvements in homology search methodology and functional predictions are being complemented by the increase in the volume of sequence data with which comparative analyses can be performed. The experimental methods needed for investigation of gene function and expression in a variety of model systems of infection continue to develop. The identification of surface-exposed microbial structures and their conservation in natural populations of pathogenic species offers prospects for developing novel vaccines. A major challenge is the development of efficient screening methods to select the most promising candidates, such as immunisation with DNA.     

Novel metal sites in protein structures

Recently, the three-dimensional structures of several novel metalloenzymes have been solved. Of special interest are those containing uncommon and/or not well characterized metals such as molybdenum, tungsten, nickel, vanadium and cobalt. Modulated by the protein environment, the specific properties of these metals and of special metal-binding cofactors such as siroheme and topa quinone are used to catalyze a vast array of fascinating reactions.