Renu Batra

Molecular mechanism for dimerization to regulate the catalytic activity of human cytomegalovirus protease.

Biochemical studies indicate that dimerization is required for the catalytic activity of herpesvirus proteases, whereas structural studies show a complete active site in each monomer, away from the dimer interface. Here we report kinetic, biophysical and crystallographic characterizations of structure-based mutants in the dimer interface of human cytomegalovirus (HCMV) protease. Such mutations can produce a 1,700-fold reduction in the kcat while having minimal effects on the Km. Dimer stability is not affected by these mutations, suggesting that dimerization itself is insufficient for activity. There are large changes in monomer conformation and dimer organization of the apo S225Y mutant enzyme. However, binding of an activated peptidomimetic inhibitor induced a conformation remarkably similar to the wild type protease. Our studies suggest that appropriate dimer formation may be required to indirectly stabilize the protease oxyanion hole, revealing a novel mechanism for dimerization to regulate enzyme activity.

Functional characterisation of Dictyostelium myosin II with conserved tryptophanyl residue 501 mutated to tyrosine

Abstract We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.

Crystal structure of MTH169, a crucial component of phosphoribosylformylglycinamidine synthetase

Introduction Phosphoribosylformylglycinamidine (FGAM) synthetase (EC 6.3.5.3) catalyses the reaction 5 -phosphoribosylformylglycinamide (FGAR) glutamine ATP 7 FGAM glutamate ADP Pi, the fourth step in the de novo purine biosynthetic pathway. In eukaryotes and many bacterial systems (including Escherichia coli and Salmonella typhimurium), the FGAM synthetase is encoded by a large protein with an N-terminal ATPase domain and a C-terminal glutamine-binding domain. In archaeal and other bacterial systems, however, FGAM synthetase is encoded by separate genes, making it a multisubunit (rather than multidomain) enzyme. For example, in Bacillus subtilis, the purL protein is homologous to the ATPase domain, whereas the purQ protein is homologous to the glutamine-binding domain of the singlechain FGAM synthetases. The purL and purQ genes are part of the pur operon in B. subtilis, which encodes 11 of the 12 enzymes in the purine biosynthetic pathway. The genetic studies also identified an open reading frame (ORF) of 84 amino acids in this operon, now known as purS, which is conserved in a large group of Gram-positive bacteria and methanogenic archaea (Fig. 1).Recent studies showed that disruption of the purS gene in B. subtilis resulted in a purineauxotrophic phenotype, due to defective FGAM synthetase activity. Therefore, the purS protein appears to be required for the function of the purL and purQ subunits of the FGAM synthetase, but the molecular mechanism for the functional role of purS is currently not known. We recently initiated a prototype structural genomics effort that focused on non-membrane proteins in the proteome of the thermophilic archaeon Methanobacterium thermoautotrophicum (Mth). One of the proteins selected for this study is MTH169, which shares 29% amino acid sequence identity with that of purS in B. subtilis. The Mth proteome also contains proteins that are homologous to the purL and purQ subunits of B. subtilis. MTH169 is, therefore, the likely purS ortholog in Mth. We will use the names purS and MTH169 interchangeably here. In addition, for ease of discussion, all purS proteins are numbered according to the sequence of MTH169 (Fig. 1). The crystal structure of MTH169 (purS) has been determined at 2.56 Å resolution (Table I)and deposited at the Protein Data Bank (entry 1GTD). This 84-residue protein forms a tetramer, each subunit of which contains welldefined secondary structure elements, including a threestranded anti-parallel -sheet ( 1 through 3) and two -helices ( A and B) [Fig. 2(A)]. Helix B covers part of one face of the -sheet, forming the hydrophobic core of the structure. Residues in this core are primarily on strand 1 and helix B [Fig. 2(A)], and are generally conserved among the family of purS proteins (Fig. 1). On the other hand, residues on the other face of the -sheet are weaklyconserved and are mostly hydrophilic or charged in nature. Helix A extends away from the -sheet structure [Fig. 2(A)]. Such a conformation is probably unstable for the molecule in the monomeric state, but these residues are stabilized by quaternary interactions in the tetramer of MTH169. There are two molecules of MTH169 in the crystallographic asymmetric unit, which form a noncrystallographic dimer [Fig. 2(B)]. The dimer is situated near a crystallographic twofold symmetry axis, and this produces a tetramer of MTH169 in the crystal [Fig. 2(C)]. Within each monomer, about 1,100 Å and 650 Å of the surface area is buried at the dimer and tetramer interface, respectively, suggesting that the dimer interface is more extensive than the tetramer interface. Structural searches against the Protein Data Bank, with the program Dali, showed that there are other structures with similar backbone folds, but none of them are identical to MTH169. All the structures identified by Dali have a four-stranded anti-parallel -sheet, most frequently with the extra strand inserted between A and 2 and located next to 2 of MTH169. Moreover, the A

Structural Studies of Herpesvirus Proteases

Structural studies of herpesvirus proteases establish that they belong to a new class of serine proteases and contain a novel Ser-His-His catalytic triad. Peptidomimetic inhibitors bind to the protease by forming an anti-parallel beeta-sheet with the enzyme. There are large conformational changes in the protease upon inhibitor binding, indicating that the protease is an induced-fit enzyme. Further studies are needed to understand the molecular basis for the dimerization requirement of the protease.