Structures of respiratory syncytial virus nucleocapsid protein from two crystal forms: details of potential packing interactions in the native helical form.

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.

Crystallization and preliminary X-ray analysis of the human respiratory syncytial virus nucleocapsid protein.

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.

Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium.

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.

Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog.

The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.

Structural basis for the resilience of efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase.

BACKGROUND: Efavirenz is a second-generation non-nucleoside inhibitor of HIV-1 reverse transcriptase (RT) that has recently been approved for use against HIV-1 infection. Compared with first-generation drugs such as nevirapine, efavirenz shows greater resilience to drug resistance mutations within HIV-1 RT. In order to understand the basis for this resilience at the molecular level and to help the design of further-improved anti-AIDS drugs, we have determined crystal structures of efavirenz and nevirapine with wild-type RT and the clinically important K103N mutant. RESULTS: The relatively compact efavirenz molecule binds, as expected, within the non-nucleoside inhibitor binding pocket of RT. There are significant rearrangements of the drug binding site within the mutant RT compared with the wild-type enzyme. These changes, which lead to the repositioning of the inhibitor, are not seen in the interaction with the first-generation drug nevirapine. CONCLUSIONS: The repositioning of efavirenz within the drug binding pocket of the mutant RT, together with conformational rearrangements in the protein, could represent a general mechanism whereby certain second-generation non-nucleoside inhibitors are able to reduce the effect of drug-resistance mutations on binding potency.

The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design.

BACKGROUND: HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS: The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS: Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.

The structure of Pneumocystis carinii dihydrofolate reductase to 1.9 A resolution.

BACKGROUND: The fungal pathogen Pneumocystis carinii causes a pneumonia which is an opportunistic infection of AIDS patients. Current therapy includes the dihydrofolate reductase (DHFR) inhibitor trimethoprim which is selective but only a relatively weak inhibitor of the enzyme for P. carinii. Determination of the three-dimensional structure of the enzyme should form the basis for design of more potent and selective therapeutic agents for treatment of the disease. RESULTS: The structure of P. carinii DHFR in complex with reduced nicotinamide adenine dinucleotide phosphate and trimethoprim has accordingly been solved by X-ray crystallography. The structure of the ternary complex has been refined at 1.86 A resolution (R = 0.181). A similar ternary complex with piritrexim (which is a tighter binding, but less selective inhibitor) has also been solved, as has the binary complex holoenzyme, both at 2.5 A resolution. CONCLUSIONS: These structures show how two drugs interact with a fungal DHFR. A comparison of the three-dimensional structure of this relatively large DHFR with bacterial or mammalian enzyme-inhibitor complexes determined previously highlights some additional secondary structure elements in this particular enzyme species. These comparisons provide further insight into the principles governing DHFR-inhibitor interaction, in which the volume of the active site appears to determine the strength of inhibitor binding.

High-resolution structures reveal details of domain closure and "half-of-sites-reactivity" in Escherichia coli aspartate beta-semialdehyde dehydrogenase.

Two high-resolution structures have been determined for Eschericia coli aspartate beta-semialdehyde dehydrogenase (ecASADH), an enzyme of the aspartate biosynthetic pathway, which is a potential target for novel antimicrobial drugs. Both ASADH structures were of the open form and were refined to 1.95 A and 1.6 A resolution, allowing a more detailed comparison with the closed form of the enzyme than previously possible. A more complex scheme for domain closure is apparent with the subunit being split into two further sub-domains with relative motions about three hinge axes. Analysis of hinge data and torsion-angle difference plots is combined to allow the proposal of a detailed structural mechanism for ecASADH domain closure. Additionally, asymmetric distortions of individual subunits are identified, which form the basis for the previously reported "half-of-the-sites reactivity" (HOSR). A putative explanation of this arrangement is also presented, suggesting the HOSR system may provide a means for ecASADH to offset the energy required to remobilise flexible loops at the end of the reaction cycle, and hence avoid falling into an energy minimum.

Ligand-induced conformational changes and a mechanism for domain closure in Aspergillus nidulans dehydroquinate synthase.

In order to investigate systematically substrate and cofactor-induced conformational changes in the enzyme dehydroquinate synthase (DHQS), eight structures representing a series of differently liganded states have been determined in a total of six crystal forms. DHQS in the absence of the substrate analogue carbaphosphonate, either unliganded or in the presence of NAD or ADP, is in an open form where a relative rotation of 11-13 degrees between N and C-terminal domains occurs. Analysis of torsion angle difference plots between sets of structures reveals eight rearrangements that appear relevant to domain closure and a further six related to crystal packing. Overlapping 21 different copies of the individual N and C-terminal DHQS domains further reveals a series of pivot points about which these movements occur and illustrates the way in which widely separated secondary structure elements are mechanically inter-linked to form "composite elements", which propagate structural changes across large distances. This analysis has provided insight into the basis of DHQS ligand-initiated domain closure and gives rise to the proposal of an ordered sequence of events involving substrate binding, and local rearrangements within the active site that are propagated to the hinge regions, leading to closure of the active-site cleft.

Crystal structures of HIV-1 reverse transcriptases mutated at codons 100, 106 and 108 and mechanisms of resistance to non-nucleoside inhibitors.

Leu100Ile, Val106Ala and Val108Ile are mutations in HIV-1 reverse transcriptase (RT) that are observed in the clinic and give rise to resistance to certain non-nucleoside inhibitors (NNRTIs) including the first-generation drug nevirapine. In order to investigate structural mechanisms of resistance for different NNRTI classes we have determined six crystal structures of mutant RT-inhibitor complexes. Val108 does not have direct contact with nevirapine in wild-type RT and in the RT(Val108Ile) complex the biggest change observed is at the distally positioned Tyr181 which is > 8 A from the mutation site. Thus in contrast to most NNRTI resistance mutations RT(Val108Ile) appears to act via an indirect mechanism which in this case is through alterations of the ring stacking interactions of the drug particularly with Tyr181. Shifts in side-chain and inhibitor positions compared to wild-type RT are observed in complexes of nevirapine and the second-generation NNRTI UC-781 with RT(Leu100Ile) and RT(Val106Ala), leading to perturbations in inhibitor contacts with Tyr181 and Tyr188. Such perturbations are likely to be a factor contributing to the greater loss of binding for nevirapine compared to UC-781 as, in the former case, a larger proportion of binding energy is derived from aromatic ring stacking of the inhibitor with the tyrosine side-chains. The differing resistance profiles of first and second generation NNRTIs for other drug resistance mutations in RT may also be in part due to this indirect mechanism.

Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second generation non-nucleoside inhibitors.

Mutations at either Tyr181 or Tyr188 within HIV-1 reverse transcriptase (RT) give high level resistance to many first generation non-nucleoside inhibitors (NNRTIs) such as the anti-AIDS drug nevirapine. By comparison second generation inhibitors, for instance the drug efavirenz, show much greater resilience to these mutations. In order to understand the structural basis for these differences we have determined a series of seven crystal structures of mutant RTs in complexes with first and second generation NNRTIs as well as one example of an unliganded mutant RT. These are Tyr181Cys RT (TNK-651) to 2.4 A, Tyr181Cys RT (efavirenz) to 2.6 A, Tyr181Cys RT (nevirapine) to 3.0 A, Tyr181Cys RT (PETT-2) to 3.0 A, Tyr188Cys RT (nevirapine) to 2.6 A, Tyr188Cys RT (UC-781) to 2.6 A and Tyr188Cys RT (unliganded) to 2.8 A resolution. In the two previously published structures of HIV-1 reverse transcriptase with mutations at 181 or 188 no side-chain electron density was observed within the p66 subunit (which contains the inhibitor binding pocket) for the mutated residues. In contrast the mutated side-chains can be seen in the NNRTI pocket for all seven structures reported here, eliminating the possibility that disordering contributes to the mechanism of resistance. In the case of the second generation compounds efavirenz with Tyr181Cys RT and UC-781 with Tyr188Cys RT there are only small rearrangements of either inhibitor within the binding site compared to wild-type RT and also for the first generation compounds TNK-651, PETT-2 and nevirapine with Tyr181Cys RT. For nevirapine with the Tyr188Cys RT there is however a more substantial movement of the drug molecule. We conclude that protein conformational changes and rearrangements of drug molecules within the mutated sites are not general features of these particular inhibitor/mutant combinations. The main contribution to drug resistance for Tyr181Cys and Tyr188Cys RT mutations is the loss of aromatic ring stacking interactions for first generation compounds, providing a simple explanation for the resilience of second generation NNRTIs, as such interactions make much less significant contribution to their binding.

Comparison of ligand-induced conformational changes and domain closure mechanisms, between prokaryotic and eukaryotic dehydroquinate synthases.

Dehydroquinate synthase (DHQS) is a potential target for the development of novel broad-spectrum antimicrobial drugs, active against both prokaryotes and lower eukaryotes. Structures have been reported for Aspergillus nidulans DHQS (AnDHQS) in complexes with a range of ligands. Analysis of these AnDHQS structures showed that a large-scale domain movement occurs during the normal catalytic cycle, with a complex series of structural elements propagating substrate binding-induced conformational changes away from the active site to distal locations. Compared to corresponding fungal enzymes, DHQS from bacterial species are both mono-functional and significantly smaller. We have therefore determined the structure of Staphylococcus aureus DHQS (SaDHQS) in five liganded states, allowing comparison of ligand-induced conformational changes and mechanisms of domain closure between fungal and bacterial enzymes. This comparative analysis shows that substrate binding initiates a large-scale domain closure in both species' DHQS and that the active site stereochemistry, of the catalytically competent closed-form enzyme thus produced, is also highly conserved. However, comparison of AnDHQS and SaDHQS open-form structures, and analysis of the putative dynamic processes by which the transition to the closed-form states are made, shows a far lower degree of similarity, indicating a significant structural divergence. As a result, both the nature of the propagation of conformational change and the mechanical systems involved in this propagation are quite different between the DHQSs from the two species.

Preliminary crystallographic data for Pneumocystis carinii dihydrofolate reductase.

Dihydrofolate reductase from Pneumocystis carinii has been crystallized in a form suitable for high resolution X-ray diffraction studies. Recombinant enzyme that had been refolded following solubilization in guanidinium hydrochloride was crystallized as both a ternary complex with the cofactor NADPH and the inhibitor trimethoprim as well as a binary complex with NADPH. The two types of complex crystallized isomorphously from polyethylene glycol using sitting-drop vapour diffusion. The crystals were of space group P2(1) with unit cell parameters, a = 69.9 A, b = 43.6 A, c = 37.6 A, beta = 117.7 degrees, with one molecule per asymmetric unit. The crystals diffracted to 1.8 A resolution.

Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution.

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.

The multifunctional folic acid synthesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase.

We describe the cloning of a multifunctional folic acid synthesis (fas) gene from Pneumocystis carinii. The nucleotide sequence contains an open reading frame interrupted by three introns, that encodes a protein of 740 amino acids with an Mr of 97,278. The predicted Fas protein has homology to two enzyme domains, dihydropteroate synthase and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, both of which are involved in folate synthesis, and at least one other region of unknown function.

Reduction in potency and reversal of left-shifting activity of BW12C with the major and minor components of chicken hemoglobin.

The effects of the left-shifting, anti-sickling compound BW12C (5-(2-formyl-3-hydroxyphenoxy)pentanoic acid) on the oxygen saturation curve of whole chicken blood and the isolated major (AII) and minor (AI) components of chicken hemoglobin have been studied. The results support the postulated major binding mode for BW12C to human hemoglobin of bridging between the alpha-chain terminal amino groups in the oxy conformation with an important hydrophobic component contributed mainly by Pro 77 alpha residues. In chicken AII (Pro 77 alpha----Ser) BW12C still left-shifts at high concentrations but its potency is greatly reduced (at least 10-fold). In chicken AI (Pro 77 alpha----Ser and Val 1 alpha----Met) BW12C is a right-shifter with a potency comparable to that of 2,3-diphosphoglycerate suggesting that binding at the beta-chain termini in the deoxy conformation is now dominant with alpha-chain binding no longer significant.

Crystallographic investigation of the cooperative interaction between trimethoprim, reduced cofactor and dihydrofolate reductase.

The structure of the complex between E. coli (RT500) form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X-ray crystallography. The inhibitor and cofactor are in mutual contact. A flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence. By contrast, the inhibitor conformation is little changed with NADPH present. We discuss these observations with regard to the mutually cooperative binding of these ligands to the protein, and to the associated enhancement of inhibitory selectivity shown by trimethoprim for bacterial as opposed to vertebrate enzyme.

Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope.

The C-termini of p66 and p51 forms of HIV-1 reverse transcriptase have been engineered to contain a Glu-Glu-Phe sequence recognized by a monoclonal antibody to alpha-tubulin, YL1/2. Mutated RTs were purified in a single step using peptide elution from columns of immobilized YL1/2. The known sequence requirements of the YL1/2 epitope are consistent with protein eluting from the column with an intact C-terminus. Kinetic parameters of these mutated RTs are essentially unchanged from wild-type enzyme. The p15 RNaseH domain has been purified using this method and shown to have low enzyme activity compared to the parental p66 subunit.

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme.

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 A resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

2.0 A X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue.

The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a betaalpha betabeta alphabeta motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents.