Venugopal Dhanaraj

Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d.

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19INK4d protein has been determined at 1.9 Å resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

A six-stranded double-psi β barrel is shared by several protein superfamilies

Abstract Background: Six-stranded β barrels with a pseudo-twofold axis are found in several proteins. One group comprises a Greek-key structure with all strands antiparallel; an example is the N-terminal domain of ferredoxin reductase. Others involve parallel strands forming two psi structures (the double-psi β barrel). A recently discovered example of the latter class is aspartate-α-decarboxylase (ADC) from Escherichia coli , a pyruvoyl-dependent tetrameric enzyme involved in the synthesis of pantothenate. Results: Visual inspection and automated database searches identified the six-stranded double-psi β barrel in ADC, Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase, E. coli formate dehydrogenase H (FDH H ), the plant defense protein barwin, Humicola insolens endoglucanase V (EGV) and, with a circular permutation, in the aspartic proteinases. Structure-based sequence alignments revealed several interactions including hydrophobic contacts or sidechain–mainchain hydrogen bonds that position the middle β strand under a psi loop, which may significantly contribute to stabilizing the fold. The identification of key interactions allowed the filtering of weak sequence similarities to some of these proteins, which had been detected by sequence database searches. This led to the prediction of the double-psi β-barrel domain in several families of proteins in eukaryotes and archaea. Conclusions: The structure comparison and clustering study of double-psi β barrels suggests that there could be a common homodimeric ancestor to ADC, FDH H and DMSO reductase, and also to barwin and EGV. There are other protein families with unknown structure that are likely to adopt the same fold. In the known structures, the protein active sites cluster around the psi loop, indicating that its rigidity, protrusion and free mainchain functional groups may be well suited to providing a framework for catalysis.

The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily.

BACKGROUND Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction. RESULTS We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E. coli. The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution. The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. CONCLUSIONS The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex.

Protein-protein interactions in receptor activation and intracellular signalling.

Abstract We review here signalling complexes that we have defined using X-ray analysis in our laboratory. They include growth factors and their receptors: nerve growth factor (NGF) and its hetero-hexameric 7S NGF storage complex, hepatocyte growth factor/scatter factor (HGF/SF) NK1 dimers and fibroblast growth factor (FGF1) in complex with its receptor (FGFR2) ectodomain and heparin. We also review our recent structural studies on intracellular signalling complexes, focusing on phosducin transducin Gβγ, CK2 protein kinase and its complexes, and the cyclin D-dependent kinase, Cdk6, bound to the cell cycle inhibitor p19INK4d. Comparing the structures of these complexes with others we show that the surface area buried in signalling interactions does not always give a good indication of the strength of the interactions. We show that conformational changes are often important in complexes with intermediate buried surface areas of 1500 to 2000 Å2, such as Cdk6 INK4 interactions. Some interactions involve recognition of continuous epitopes, where there is no necessity for a tertiary structure and very often the binding conformation is induced during the process of interaction, for example phosducin binding to the βγ subunits (Gtβγ) of the heterotrimeric G protein transducin.

Structure of E. coli Ketopantoate Hydroxymethyl Transferase Complexed with Ketopantoate and Mg2+, Solved by Locating 160 Selenomethionine Sites

We report the crystal structure of E. coli ketopantoate hydroxymethyltransferase (KPHMT) at 1.9 A resolution, in complex with its product, ketopantoate. KPHMT catalyzes the first step in the biosynthesis of pantothenate (vitamin B(5)), the precursor of coenzyme A and the acyl carrier protein cofactor. The structure of the decameric enzyme was solved by multiwavelength anomalous dispersion to locate 160 selenomethionine sites and phase 560 kDa of protein, making it the largest structure solved by this approach. KPHMT adopts the (betaalpha)(8) barrel fold and is a member of the phosphoenolpyruvate/pyruvate superfamily. The active site contains a ketopantoate bidentately coordinated to Mg(2+). Similar binding is likely for the substrate, alpha-ketoisovalerate, orienting the C3 for deprotonation.

The Aspartic Proteinases

Research into the aspartic proteinases has had a rich and diverse history. It began with studies of the digestive juices of man and nepenthes using somewhat primitive technologies. It had a renaissance of biochemical characterisation, followed by a classical period when sequences were defined and catalytic activity identified. It is now very much under the influence of the structural school. Thus, in many ways its history parallels that of painting. And like painting it is now thoroughly commercialised, a mixed blessing which nevertheless keeps many of its practioners well above the breadline.

Structure of the regulatory subunit of CK2 in the presence of a p21WAF1 peptide demonstrates flexibility of the acidic loop

A truncated form of the regulatory subunit of the protein kinase CK2beta (residues 1-178) has been crystallized in the presence of a fragment of the cyclin-dependent kinase inhibitor p21WAF1 (residues 46-65) and the structure solved at 2.9 A resolution by molecular replacement. The core of the CK2beta dimer shows a high structural similarity with that identified in previous structural analyses of the dimer and the holoenzyme. However, the electron density corresponding to the substrate-binding acidic loop (residues 55-64) indicates two conformations that differ from that of the holoenzyme structure [Niefind et al. (2001), EMBO J. 20, 5320-5331]. Difference electron density near the dimerization region in each of the eight protomers in the asymmetric unit is attributed to between one and eight amino-acid residues of a complexed fragment of p21WAF1. This binding site corresponds to the solvent-accessible part of the conserved zinc-finger motif.

Aspartic proteinases: The structures and functions of a versatile superfamily of enzymes

This paper reviews the structure and function of monomeric eukaryotic aspartic proteinases and their inhibitors, including recent analyses of the sequences and the three-dimensional structural models of the plant aspartic proteinases, which contain a very large inserted domain that is homologous to saposins. The three-dimensional structures of renins, cathepsin D and cathepsin E complexed with inhibitors are described. These have provided an understanding of the relation between structure, catalysis and specificity useful for drug design. Finally, studies are presented of homologues of aspartic proteinases which are found during pregnancy in livestock; these have lost the catalytic residues characteristic of active enzymes, although they have the capacity to bind peptides.

Protein three-dimensional structure and molecular recognition: a story of soft locks and keys

Abstract One hundred years ago Emil Fischer proposed a descriptive but provocative analogy for molecular recognition: the lock and key hypothesis. At a time when little was known of the molecular structures of even the relatively simple substrates of enzymes, let alone the complex structures of proteins, this gave an extraordinarily useful visual image of enzyme action. Similar recognition processes, such as antigen-antibody, hormone or growth factor-receptor, lectin-sugar, repressor-DNA and so on, have since been identified in other classes of proteins. Can the Fischer hypothesis be applied to these systems? Has the hypothesis stood the test of time? In this paper, we examine the crystal structures of proteins complexed with their ligand molecules: the pentraxins bound to carbohydrate, several aspartic proteinases complexed with inhibitors, the SH3 domains bound to proline-rich peptide motifs, the periplasmic binding proteins and growth factor systems bound to cell surface receptors. We discuss the modes of binding in terms of surface rigidity, charge and shape complementarity. Such recognition processes are often accompanied by distinct conformational changes at the binding site. The ligand selectivity demonstrated in these systems supports a “soft” lock-and-key hypothesis.

N-ethylmaleimide-sensitive fusion protein (NSF) and CDC48 confirmed as members of the double-psi β-barrel aspartate decarboxylase/formate dehydrogenase family

Mays main criticism is that the topology of our dendrogram (Figure 4 of Castillo et al. [1xA six-stranded double-psi β barrel is shared by several protein superfamilies. Castillo, R.M., Mizuguchi, K., Dhanaraj, V., Albert, A., Blundell, T.L., and Murzin, A.G. Structure. 1999; 7: 227–236Abstract | Full Text | Full Text PDF | PubMed | Scopus (71)See all References][1]) for the individual halves of double-psi β barrels is unstable. This is a fair point and his jackknife test is a useful way to detect the problem, although the unstable nature of the tree could be suspected even from the published figure alone, as some of the branch lengths are very short. It should be noted here (and it is not clear whether May recognizes this point) that our dendrogram was neither a phylogenetic tree, nor was it drawn even to examine the evolutionary relationships between these structures. This is a simple two-dimensional representation of the structural similarities defined in a particular way, and it can be as misleading as any other two-dimensional representation of distance data.As for Mays criticism that we used this dendrogram as support for an evolutionary scenario, we did not explicitly propose the existence of a common homodimeric ancestor to all five proteins aspartate decarboxylase (ADC; 1aw8), dimethylsulphoxide reductase (DMSO reductase; 1cxs), formate dehydrogenase H (FDHH; 1fdo), barwin (1bw4) and endoglucanase V (EGV; 2eng). We stated that there could be a common homodimeric ancestor to ADC, FDHH and DMSO reductase, and also to barwin and EGV”. The existence of a single homodimeric ancestor to all five protein families was certainly not a major conclusion in our paper.On the other hand, an evolutionary relationship between the ADC and FDHH families was one of our main conclusions and it should be emphasized that this was supported by an independent analysis summarized in Figure 7 of [1xA six-stranded double-psi β barrel is shared by several protein superfamilies. Castillo, R.M., Mizuguchi, K., Dhanaraj, V., Albert, A., Blundell, T.L., and Murzin, A.G. Structure. 1999; 7: 227–236Abstract | Full Text | Full Text PDF | PubMed | Scopus (71)See all References][1]. We examined the local structural environments in a conserved region, a proposed determinant of the double-psi topology, in the N-terminal half of the ADC and FDHH structures. We then identified key positions that can accommodate only certain types of amino acids. On the basis of this knowledge, we predicted that several other protein families might adopt the same fold and share the common ancestor. One of the families we identified was CDC48; a domain of the double-psi β-barrel fold was predicted in an N-terminal region of CDC48. Also, the sequence of this domain was found to contain two copies of the topological determinant, one in the N-terminal half and the other in the symmetry-related C-terminal half. It was, therefore, suggested that the predicted pseudo-symmetrical structure of the CDC48 domain might approximate the symmetrical structure of a hypothetical homodimeric ancestor to the ADC, FDHH and related families.In fact, new experimental evidence is now available to support our hypothesis. A new example of the double-psi β-barrel fold has been discovered recently in the structure of the N-terminal fragment of N-ethylmaleimide-sensitive fusion protein (NSF) [2xCrystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein. May, A.P., Misura, K.M.S., Whiteheart, S.W., and Weis, W.I. Nat. Cell Biol. 1999; 1: 175–182Crossref | PubMedSee all References][2]. NSF consists of three domains: an N-terminal domain (NSF-N) and a tandem repeat of two domains belonging to the AAA family of ATPases. A similar tandem repeat of two AAA domains is also found in CDC48. The probable distant homology between the NSF and CDC48 families is further supported by their functional similarity, but their apparent sequence similarity is confined to the two AAA domains [3xAn abundant and ubiquitous homo-oligomeric ring-shaped ATPase particle related to the putative vesicle fusion proteins Sec 18p and NSF. Peters, J.-M., Walsh, M.J., and Franke, W.W. EMBO J. 1990; 9: 1757–1767PubMedSee all References][3]. May et al. produced a sequence alignment of NSF-N and related domains and, based on the conservation of key residues, they concluded that the homology between CDC48 and NSF extends through the entire sequences. Moreover, the sequence alignment of NSF-N and CDC48, combined with our original alignment in Figure 7, now allowed us to align the sequence of NSF-N with those of other double-psi β-barrel structures. This sequence alignment is virtually identical to a direct structure-based sequence alignment of NSF-N (taken from PDB entry 1qdn), ADC and FDHH (Figure 1Figure 1). In the C-terminal half of the NSF-N double-psi β barrel, there is the same topological determinant that we discovered in the symmetry-related position in the N-terminal half of the ADC and FDHH barrels. Thus the NSF-N double-psi β barrel might have descended from the same hypothetical homodimeric ancestor through a different evolutionary route. This analysis strongly suggests that an evolutionary scenario linking the ADC and FDHH families is indeed the case.Figure 1Stereoview of the double-psi β-barrel domains from NSF-N (PDB entry 1qdn; blue), ADC (1aw8; green) and FDHH (1fdo; red) with their common topological determinant superimposed. This determinant consists of contiguous sequence of some 25 residues (residues 59–82 of NSF-N, 25–48 of ADC and 607–630 of FDHH; thick lines) forming a β-α-turn-β structure. The superposition of these regions in the three structures gives root mean square deviations (rmsds) of less than 1 A for all mainchain atoms. The rest of the common structural core (about 30 additional residues) also overlays well in this superposition with pairwise rmsd from 1.7 to 2.3 A. It should be noted that in this superposition, the orientation of the NSF-N domain is related to those of the other two structures by the pseudo-twofold symmetry axis of the double-psi β-barrel fold.View Large Image | View Hi-Res Image | Download PowerPoint Slide