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Respiratory syncytial virus (RSV) is a frequent cause of respiratory illness in infants, but there is currently no vaccine nor effective drug treatment against this virus. The RSV RNA genome is encapsidated and protected by a nucleocapsid protein; this RNA-nucleocapsid complex serves as a template for viral replication. Interest in the nucleocapsid protein has increased owing to its recent identification as the target site for novel anti-RSV compounds. The crystal structure of human respiratory syncytial virus nucleocapsid (HRSVN) was determined to 3.6 Å resolution from two crystal forms belonging to space groups P2(1)2(1)2(1) and P1, with one and four decameric rings per asymmetric unit, respectively. In contrast to a previous structure of HRSVN, the addition of phosphoprotein was not required to obtain diffraction-quality crystals. The HRSVN structures reported here, although similar to the recently published structure, present different molecular packing which may have some biological implications. The positions of the monomers are slightly shifted in the decamer, confirming the adaptability of the ring structure. The details of the inter-ring contacts in one crystal form revealed here suggest a basis for helical packing and that the stabilization of native HRSVN is via mainly ionic interactions., (© 2011 International Union of Crystallography. All rights reserved.)

Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and approximately 5 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 A, and space group P2(1), with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 A, beta = 90.1 degrees , respectively. For unlabelled HRSVN, only crystals belonging to space group P2(1) were obtained that diffracted to 3.6 A. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.

The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

Nucleotide monophosphate kinases (NMPKs) are potential antimicrobial drug targets owing to their role in supplying DNA and RNA precursors. The present work reports the crystal structure of Staphylococcus aureus guanylate monophosphate kinase (SaGMK) at 1.9 A resolution. The structure shows that unlike most GMKs SaGMK is dimeric, confirming the role of the extended C-terminus in dimer formation as first observed for Escherichia coli GMK (EcGMK). One of the two SaGMK dimers within the crystal asymmetric unit has two monomers in different conformations: an open form with a bound sulfate ion (mimicking the beta-phosphate of ATP) and a closed form with bound GMP and sulfate ion. GMP-induced domain movements in SaGMK can thus be defined by comparison of these conformational states. Like other GMKs, the binding of GMP firstly triggers a partial closure of the enzyme, diminishing the distance between the GMP-binding and ATP-binding sites. In addition, the closed structure shows the presence of a potassium ion in contact with the guanine ring of GMP. The potassium ion appears to form an integral part of the GMP-binding site, as the Tyr36 side chain has significantly moved to form a metal ion-ligand coordination involving the lone pair of the side-chain O atom. The potassium-binding site might also be exploited in the design of novel inhibitors.

The crystal structure of Staphylococcus aureus cytidine monophosphate kinase (CMK) in complex with cytidine 5'-monophosphate (CMP) has been determined at 2.3 angstroms resolution. The active site reveals novel features when compared with two orthologues of known structure. Compared with the Streptococcus pneumoniae CMK solution structure of the enzyme alone, S. aureus CMK adopts a more closed conformation, with the NMP-binding domain rotating by approximately 16 degrees towards the central pocket of the molecule, thereby assembling the active site. Comparing Escherichia coli and S. aureus CMK-CMP complex structures reveals differences within the active site, including a previously unreported indirect interaction of CMP with Asp33, the replacement of a serine residue involved in the binding of CDP by Ala12 in S. aureus CMK and an additional sulfate ion in the E. coli CMK active site. The detailed understanding of the stereochemistry of CMP binding to CMK will assist in the design of novel inhibitors of the enzyme. Inhibitors are required to treat the widespread hospital infection methicillin-resistant S. aureus (MRSA), currently a major public health concern.

Crystallization of Aspergillus nidulans 3-dehydroquinate synthase (DHQS), following turnover of the enzyme by addition of the substrate DAHP, gave a new crystal form (form J). Although the crystals have dimensions of only 50 x 20 x 5 micro m, they are well ordered, diffracting to 1.7 A. The space group is C222(1), with unit-cell parameters a = 90.0, b = 103.7, c = 177.4 A. Structure determination and refinement to R = 0.19 (R(free) = 0.25) shows the DHQS is in the 'open' form with the substrate site unoccupied but with some loop regions perturbed. Previous crystals of open-form DHQS only diffracted to 2.5 A resolution. The use of enzyme turnover may be applicable in other systems in attempts to improve crystal quality.

The NmrA repressor protein of Aspergillus nidulans was overproduced in Escherichia coli and purified to homogeneity. Gel-exclusion chromatography showed that NmrA was monomeric in solution under the buffer conditions used. The protein was crystallized in three forms, belonging to trigonal, monoclinic and hexagonal space groups. Two of these crystal forms (A and B) diffract to high resolution and thus appear suitable for structure determination. Crystal form A belongs to space group P3((1))21, with unit-cell parameters a = b = 76.8, c = 104.9 A. Crystal form B belongs to space group C2, with unit-cell parameters a = 148.8, b = 64.3, c = 110.2 A, beta = 121.8 degrees.

Extensive crystallization trials of Aspergillus nidulans dehydroquinate synthase, a potential novel target for antimicrobial drugs, in complexes with different ligands have resulted in the identification of nine crystal forms. Crystals of unliganded DHQS, binary complexes with either the substrate analogue, carbaphosphonate or the cofactor NADH, as well as the ternary DHQS-carbaphosphonate-cofactor complex, were obtained. The ternary complex crystallizes from ammonium sulfate and CoCl(2) in space group P2(1)2(1)2, with unit-cell parameters a = 133.8, b = 86.6, c = 74.9 A. The binary carbaphosphonate complex crystallizes from PEG 6000 in space group P2(1)2(1)2(1), with a = 70.0, b = 64.0, c = 197.6 A, and the binary cofactor complex crystallizes from PEG 3350 and sodium potassium tartrate in space group P2(1), with a = 83.7, b = 70.4, c = 144.3 A, beta = 89.2 degrees. DHQS in the absence of ligands crystallizes in space group P2(1), with a = 41.0, b = 68.9, c = 137.7 A, beta = 94.8 degrees. Each of these crystal forms are suitable for high-resolution structure determination. Structures of a range of DHQS-ligand complexes will be of value in the structure-based design of novel antimicrobial drugs.