Martin Noble

Active and inactive protein kinases: structural basis for regulation.

lytic groups and relief of steric blocking to allow access Protein kinases and phosphatases play pivotal roles in of substrates to the catalytic site. The activation segregulating and coordinating aspects of metabolism, ment and the control of its conformation via phosphorygene expression, cell growth, cell motility, cell differentilation plays a key role in these transformations. It can ation, and cell division. As a result, if cellular life is to be involved in recognition of regulatory subunits, in aufunction in an orderly manner, the switching on and off toinhibition of substrate binding, and in promotion of of protein kinases and phosphatases is as crucial for the correct orientation of domains and catalytic site resitheir function as their catalytic activity. dues. This review summarizes our current understandThe total number of distinct kinase domain sequences ing of control by the activation segment based on recent available is now approaching 400 (Hardie and Hanks, structure determinations of active and inactive kinases. 1995). Multiple sequence alignments have indicated that The first observation of Thr-197 phosphorylation in all protein kinases should have similar structures, and the activation segment of cAPK was reported in 1979 this has been confirmed by recent crystal structure de(Shoji et al., 1979). Although itwas speculated that phosterminations. Conserved features have been identified phorylation at discrete sites might be of physiological in 12 subdomain regions of all protein kinases, and resiimportance in the regulation of enzyme activity, it was dues from these subdomains have been implicated in not until 1990 that mutagenesis studies indicated the essential roles in enzyme structure and function. Protein significance of this site for the recognition of the regulakinases exhibit variability in other parts of the kinase tory subunit (Levin and Zoller, 1990), and not until 1993 domain, and different kinases may contain additional that it was definitively shown that phosphorylation is domains, additional subunits, or both. These features promoted by an autocatalytic event that is crucial for allow several different mechanisms for control. activation (Steinberg et al., 1993). The crystal structure Control mechanisms that have been recognized to determination of cAPK in 1991 (Knighton et al., 1991a, date include the following: control by additional subunits 1991b) showed the structural importance of Thr-197 or domains that may function in response to second phosphorylation and demonstrated possible roles of messengers (e.g., cyclic AMP binding to the regulatory phosphorylation in promotion of activation. The strucsubunit of cyclic AMP–dependent protein kinase [cAPK], ture provided a definitive model to which other kinases Ca/calmodulin binding to calmodulin-dependent could be related. Also in 1991, both the fission yeast protein kinases, and Ca and diacyl glyerol binding to cell division control kinase (cdc2) (Gould et al., 1991) N-terminal domains of protein kinase C); control by addiand the microtubule-associated protein kinase (MAPK) tional subunits whose level of expression varies de(Payne et al., 1991) were found to be activated by phospending on the functional state of the cell (e.g., cyclin phorylation on residues that mapped to a position simiregulation of the cyclin-dependent protein kinases lar to Thr-197 in cAPK. These results showed the impor[CDKs]); control by additional domains that target the tance of this site not only as an autophosphorylation kinase to different molecules or subcellular localizations site, as in cAPK, but also as a site involved in kinase (e.g., the SH2 and SH3 domains of the Src kinases); cascade activation mechanisms. For the tyrosine kicontrol by additional domains that inhibit the kinase nases, autophosphorylation of pp60 at position Tyractivity by an autoregulatory process (e.g., myosin light 416 (now known to be in the activation segment) had chain kinase [MLCK]); and control by phosphorylation been shown in the early 1980s, and its significance for and dephosphorylation by kinases and phosphatases. control in the cellular counterpart of Src kinase was Phosphorylation of specific threonine, serine, or tyrosine established by 1987 (reviewed by Hunter, 1987). The residues may occur at a number of sites. Some of these following year, trans-autophosphorylation of the insulin are located in the N-terminal or C-terminal portions of receptor tyrosine kinase (IRK) was elaborated, and the the polypeptide chain, which lie outside the kinase dosimilarity in sequence location of some of these sites main (e.g., in Src kinase and calmodulin-dependent kito that in Src kinase and its relatives and in cAPK was nase II [CaMKII]) or on othersubunits (e.g., in phosphorynoted (reviewed by White and Kahn, 1994). As more and lase kinase [PhK]). A key aspect of regulation recognized more kinases have been discovered and sequenced and in recent years is that many protein kinases are phosfurther kinase cascades established, it is recognized phorylated on a residue(s) located in a particular segthat control by phosphorylation in the activation segment in the center of the kinase domain, which is termed ment is a property of most, but not all, protein kinases

Presenting your structures: the CCP4mg molecular-graphics software

The CCP4 molecular-graphics program now uses the Qt framework to provide a modern look and feel. There are many new features including rendering for publication-quality images and sequence alignment.

Developments in the CCP4 molecular-graphics project

Progress towards structure determination that is both high-throughput and high-value is dependent on the development of integrated and automatic tools for electron-density map interpretation and for the analysis of the resulting atomic models. Advances in map-interpretation algorithms are extending the resolution regime in which fully automatic tools can work reliably, but at present human intervention is required to interpret poor regions of macromolecular electron density, particularly where crystallographic data is only available to modest resolution [for example, I/sigma(I) < 2.0 for minimum resolution 2.5 A]. In such cases, a set of manual and semi-manual model-building molecular-graphics tools is needed. At the same time, converting the knowledge encapsulated in a molecular structure into understanding is dependent upon visualization tools, which must be able to communicate that understanding to others by means of both static and dynamic representations. CCP4 mg is a program designed to meet these needs in a way that is closely integrated with the ongoing development of CCP4 as a program suite suitable for both low- and high-intervention computational structural biology. As well as providing a carefully designed user interface to advanced algorithms of model building and analysis, CCP4 mg is intended to present a graphical toolkit to developers of novel algorithms in these fields.

The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate

The family of lipases (triacylglycerol‐acyl‐hydrolases, EC 3.1.1.3) constitutes an interesting class of enzymes because of their ability to interact with lipid‐water interfaces, their wide range of substrate specificities, and their potential industrial applications [1,2]. Here we report the first crystal structure of a bacterial lipase, from Pseudomonas glumae. The structure is formed from three domains, the largest of which contains a subset of the α/β hydrolase fold and a calcium site. Asp263, the acidic residue in the catalytic triad, has previously been mutated into an alanine with only a modest reduction in activity [3].

Cyclin dependent kinase inhibitors

Cyclin-dependent kinases are involved in diverse cellular processes that include cell cycle control, apoptosis, neuronal physiology, differentiation, and transcription. Intensive screening and drug design based on CDK/inhibitor co-crystal structures and on SAR studies have led to the identification and characterization of a large variety of chemical inhibitors of CDKs. Although they all act by competing with ATP for binding at the catalytic site of the kinase, their kinase selectivity varies greatly and remains to be studied in most cases. The requirement for CDKs in many physiological processes justifies their evaluation as potential therapeutic targets against a much larger scope of diseases than initially anticipated.Cell-cycle dysregulation is one of the cardinal characteristics of neoplastic cells. For this reason, small molecule inhibitors targeting cyclin-dependent kinases (CDKs), of which flavopiridol is a prototype, have been the focus of extensive interest in cancer therapy. In addition to inhibiting cell-cycle progression, these agents exhibit a variety of other activities, including the induction of cell death. Recently, several novel mechanisms of action have been ascribed to the CDK inhibitor flavopiridol, including interference with transcription, most likely through disruption of P-TEFb (i.e. the CDK9/cyclin T complex), and induction of apoptosis, possibly a consequence of downregulation of various anti-apoptotic proteins. It has also been observed that combining CDK inhibitors with either conventional cytotoxic drugs or novel signal transduction modulators dramatically promotes neoplastic cell death in a variety of preclinical models. Efforts are underway to uncover inhibitors that selectively target specific CDKs and to develop these as a new generation of antitumour drugs. For all of these reasons, it is likely that interest in CDK inhibitors as antineoplastic agents will continue for the foreseeable future.

The Structural Basis for Control of Eukaryotic Protein Kinases

Eukaryotic protein kinases are key regulators of cell processes. Comparison of the structures of protein kinase domains, both alone and in complexes, allows generalizations to be made about the mechanisms that regulate protein kinase activation. Protein kinases in the active state adopt a catalytically competent conformation upon binding of both the ATP and peptide substrates that has led to an understanding of the catalytic mechanism. Docking sites remote from the catalytic site are a key feature of several substrate recognition complexes. Mechanisms for kinase activation through phosphorylation, additional domains or subunits, by scaffolding proteins and by kinase dimerization are discussed.

Structures of the Cd44-Hyaluronan Complex Provide Insight Into a Fundamental Carbohydrate-Protein Interaction.

Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The molecular details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equivalent to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.

The CCP4 molecular-graphics project

This new package will provide easy-to-use access to crystallographic structure solution, model building and structure analysis. It will be possible for any developer to integrate scientific software into the system.

The structural basis for substrate recognition and control by protein kinases1

Protein kinases catalyse phospho transfer reactions from ATP to serine, threonine or tyrosine residues in target substrates and provide key mechanisms for control of cellular signalling processes. The crystal structures of 12 protein kinases are now known. These include structures of kinases in the active state in ternary complexes with ATP (or analogues) and inhibitor or peptide substrates (e.g. cyclic AMP dependent protein kinase, phosphorylase kinase and insulin receptor tyrosine kinase); kinases in both active and inactive states (e.g. CDK2/cyclin A, insulin receptor tyrosine kinase and MAPK); kinases in the active state (e.g. casein kinase 1, Lck); and kinases in inactive states (e.g. twitchin kinase, calcium calmodulin kinase 1, FGF receptor kinase, c‐Src and Hck). This paper summarises the detailed information obtained with active phosphorylase kinase ternary complex and reviews the results with reference to other kinase structures for insights into mechanisms for substrate recognition and control.

Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) fromSaccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-Å resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.