Optics Concept for a Pair of Undulator Beamlines for MX.

We describe a concept for x-ray optics to feed a pair of macromolecular crystallography (MX) beamlines which view canted undulator radiation sources in the same storage ring straight section. It can be deployed at NSLS-II and at other low-emittance third-generation synchrotron radiation sources where canted undulators are permitted, and makes the most of these sources and beamline floor space, even when the horizontal angle between the two canted undulator emissions is as little as 1-2 mrad. The concept adopts the beam-separation principles employed at the 23-ID (GM/CA-CAT) beamlines at the Advanced Photon Source (APS), wherein tandem horizontally-deflecting mirrors separate one undulator beam from the other, following monochromatization by a double-crystal monochromator. The scheme described here would, in contrast, deliver the two tunable monochromatic undulator beams to separate endstations that address rather different and somewhat complementary purposes, with further beam conditioning imposed as required. A downstream microfocusing beamline would employ dual-stage focusing for work at the micron scale and, unique to this design, switch to single stage focusing for larger beams. On the other hand, the upstream, more highly automated beamline would only employ single stage focusing.

Multiwavelength anomalous diffraction analysis at the M absorption edges of uranium.

The multiwavelength anomalous diffraction (MAD) method for phase evaluation is now widely used in macromolecular crystallography. Successful MAD structure determinations have been carried out at the K or L absorption edges of a variety of elements. In this study, we investigate the anomalous scattering properties of uranium at its M(IV) (3.326 A) and M(V) (3.490 A) edge. Fluorescence spectra showed remarkably strong anomalous scattering at these edges (f' = -70e, f" = 80e at the M(IV) edge and f' = -90e, f" = 105e at the M(V) edge), many times higher than from any anomalous scatterers used previously for MAD phasing. However, the large scattering angles and high absorption at the low energies of these edges present some difficulties not found in typical crystallographic studies. We conducted test experiments at the M(IV) edge with crystals of porcine elastase derivatized with uranyl nitrate. A four-wavelength MAD data set complete to 3.2-A Bragg spacings was collected from a single small frozen crystal. Analysis of the data yielded satisfactory phase information (average difference of (0)phi(T) - (0)phi(A) for replicated determinations is 32 degrees ) and produced an interpretable electron-density map. Our results demonstrate that it is practical to measure macromolecular diffraction data at these edges with current instrumentation and that phase information of good accuracy can be extracted from such experiments. We show that such experiments have potential for the phasing of very large macromolecular assemblages.

Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. Insight into the reaction mechanism.

PhoQ is a transmembrane histidine kinase belonging to the family of two-component signal transducing systems common in prokaryotes and lower eukaryotes. In response to changes in environmental Mg(2+) concentration, PhoQ regulates the level of phosphorylated PhoP, its cognate transcriptional response-regulator. The PhoQ cytoplasmic region comprises two independently folding domains: the histidine-containing phosphotransfer domain and the ATP-binding kinase domain. We have determined the structure of the kinase domain of Escherichia coli PhoQ complexed with the non-hydrolyzable ATP analog adenosine 5'-(beta,gamma-imino)triphosphate and Mg(2+). Nucleotide binding appears to be accompanied by conformational changes in the loop that surrounds the ATP analog (ATP-lid) and has implications for interactions with the substrate phosphotransfer domain. The high resolution (1.6 A) structure reveals a detailed view of the nucleotide-binding site, allowing us to identify potential catalytic residues. Mutagenic analyses of these residues provide new insights into the catalytic mechanism of histidine phosphorylation in the histidine kinase family. Comparison with the active site of the related GHL ATPase family reveals differences that are proposed to account for the distinct functions of these proteins.

FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions.

The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF).

Synchrotron crystallography.

The past decade has seen an explosive growth in atomic-level structures determined by X-ray crystallography. Synchrotron radiation and a number of technical advances related quite directly to its development have fueled this growth. With the most recent advances coming to be used collectively and new resources being built, the foundation is laid for a dramatic further expansion of synchrotron crystallography in the next decade. Both the high-throughput applications of structural genomics and also the challenging studies of macromolecular machinery are expected to flourish.

Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells.

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.

Energetics of the HIV gp120-CD4 binding reaction.

HIV infection is initiated by the selective interaction between the cellular receptor CD4 and gp120, the external envelope glycoprotein of the virus. We used analytical ultracentrifugation, titration calorimetry, and surface plasmon resonance biosensor analysis to characterize the assembly state, thermodynamics, and kinetics of the CD4-gp120 interaction. The binding thermodynamics were of unexpected magnitude; changes in enthalpy, entropy, and heat capacity greatly exceeded those described for typical protein-protein interactions. These unusual thermodynamic properties were observed with both intact gp120 and a deglycosylated and truncated form of gp120 protein that lacked hypervariable loops V1, V2, and V3 and segments of its N and C termini. Together with previous crystallographic studies, the large changes in heat capacity and entropy reveal that extensive structural rearrangements occur within the core of gp120 upon CD4 binding. CD spectral studies and slow kinetics of binding support this conclusion. These results indicate considerable conformational flexibility within gp120, which may relate to viral mechanisms for triggering infection and disguising conserved receptor-binding sites from the immune system.

Structure of the active core of human stem cell factor and analysis of binding to its receptor kit.

Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding portions composed of immunoglobulin-like repeats. We have determined the crystal structure of selenomethionyl soluble human SCF at 2.2 A resolution by multiwavelength anomalous diffraction phasing. SCF has the characteristic helical cytokine topology, but the structure is unique apart from core portions. The SCF dimer has a symmetric 'head-to-head' association. Using various prior observations, we have located potential Kit-binding sites on the SCF dimer. A superimposition of this dimer onto VEGF in its complex with the receptor Flt-1 places the binding sites on SCF in positions of topographical and electrostatic complementarity with the Kit counterparts of Flt-1, and a similar model can be made for the complex of PDGF with its receptor.

Structural hierarchy in erythrocruorin, the giant respiratory assemblage of annelids.

Many annelids, including the earthworm Lumbricus terrestris, have giant cooperative respiratory proteins (molecular masses greater than 3.5 million Da) freely dissolved in the blood, rather than packaged in cells. These complexes, termed either erythrocruorins or hemoglobins, are assembled from many copies of both hemoglobin subunits and nonhemoglobin or "linker" subunits. In this paper, we present the crystal structure of Lumbricus erythrocruorin at 5.5-A resolution, which reveals a remarkable hierarchical organization of 144 oxygen-binding hemoglobin subunits and 36 nonhemoglobin linker subunits. The hemoglobin chains arrange in novel dodecameric substructures. Twelve trimeric linker complexes project triple-stranded helical coiled-coil "spokes" toward the center of the complex; interdigitation of these spokes appears crucial for stabilization. The resulting complex of linker chains forms a scaffold on which twelve hemoglobin dodecamers assemble. This structure specifies the unique, self-limited assemblage of a highly cooperative single molecule.

Protein folding and unfolding by Escherichia coli chaperones and chaperonins.

The folding of proteins from their initial unstructured state to their mature form has long been known to be promoted by other proteins known as chaperones and chaperonins. Recent biochemical and structural discoveries have provided dramatic insight into how these folding proteins work. This review will discuss these findings and suggest future experimental directions.

Structural interactions of fibroblast growth factor receptor with its ligands.

Fibroblast growth factors (FGFs) effect cellular responses by binding to FGF receptors (FGFRs). FGF bound to extracellular domains on the FGFR in the presence of heparin activates the cytoplasmic receptor tyrosine kinase through autophosphorylation. We have crystallized a complex between human FGF1 and a two-domain extracellular fragment of human FGFR2. The crystal structure, determined by multiwavelength anomalous diffraction analysis of the selenomethionyl protein, is a dimeric assemblage of 1:1 ligand:receptor complexes. FGF is bound at the junction between the two domains of one FGFR, and two such units are associated through receptor:receptor and secondary ligand:receptor interfaces. Sulfate ion positions appear to mark the course of heparin binding between FGF molecules through a basic region on receptor D2 domains. This dimeric assemblage provides a structural mechanism for FGF signal transduction.

Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus.

The human immunodeficiency virus envelope glycoproteins, gp120 and gp41, function in cell entry by binding to CD4 and a chemokine receptor on the cell surface and orchestrating the direct fusion of the viral and target cell membranes. On the virion surface, three gp120 molecules associate noncovalently with the ectodomain of the gp41 trimer to form the envelope oligomer. Although an atomic-level structure of a monomeric gp120 core has been determined, the structure of the oligomer is unknown. Here, the orientation of gp120 in the oligomer is modeled by using quantifiable criteria of carbohydrate exposure, occlusion of conserved residues, and steric considerations with regard to the binding of the neutralizing antibody 17b. Applying similar modeling techniques to influenza virus hemagglutinin suggests a rotational accuracy for the oriented gp120 of better than 10 degrees. The model shows that CD4 binds obliquely, such that multiple CD4 molecules bound to the same oligomer have their membrane-spanning portions separated by at least 190 A. The chemokine receptor, in contrast, binds to a sterically restricted surface close to the trimer axis. Electrostatic analyses reveal a basic region which faces away from the virus, toward the target cell membrane, and is conserved on core gp120. The electrostatic potentials of this region are strongly influenced by the overall charge, but not the precise structure, of the third variable (V3) loop. This dependence on charge and not structure may make electrostatic interactions between this basic region and the cell difficult to target therapeutically and may also provide a means of viral escape from immune system surveillance.

Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates.

BACKGROUND: The gp120 exterior envelope glycoprotein of HIV-1 binds sequentially to CD4 and chemokine receptors on cells to initiate virus entry. During natural infection, gp120 is a primary target of the humoral immune response, and it has evolved to resist antibody-mediated neutralization. We previously reported the structure at 2.5 A of a gp120 core from the HXBc2 laboratory-adapted isolate in complex with a 2 domain fragment of CD4 and the antigen binding fragment of a human antibody. This revealed atomic details of gp120-receptor interactions and suggested multiple mechanisms of immune evasion. RESULTS: We have now extended the HXBc2 structure in P222, crystals to 2.2 A. The enhanced resolution enabled a more accurate modeling of less-well-ordered regions and provided conclusive identification of the density in the central cavity at the crux of the gp120-CD4 interaction as isopropanol from the crystallization medium. We have also determined the structure of a gp120 core from the primary clinical HIV-1 isolate, YU2, in the same ternary complex but in a C2 crystal lattice. Comparisons of HXBc2 and YU2 showed that while CD4 binding was rigid, portions of the gp120 core were conformationally flexible; overall differences were minor, with sequence changes concentrated on a surface expected to be exposed on the envelope oligomer. CONCLUSIONS: Despite dramatic antigenic differences between primary and laboratory-adapted HIV-1, the gp120 cores from these isolates are remarkably similar. Taken together with chimeric substitution and sequence analysis, this indicates that neutralization resistance is specified by quaternary interactions involving the major variable loops and thus affords a mechanism for viral adaptation. Conservation of the central cavity suggests the possibility of therapeutic inhibitors. The structures reported here extend in detail and generality our understanding of the biology of the gp120 envelope glycoprotein.

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.

Probability analysis of variational crystallization and its application to gp120, the exterior envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1).

The extensive glycosylation and conformational mobility of gp120, the envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1), pose formidable barriers for crystallization. To surmount these difficulties, we used probability analysis to determine the most effective crystallization approach and derive equations which show that a strategy, which we term variational crystallization, substantially enhances the overall probability of crystallization for gp120. Variational crystallization focuses on protein modification as opposed to crystallization screening. Multiple variants of gp120 were analyzed with an iterative cycle involving a limited set of crystallization conditions and biochemical feedback on protease sensitivity, glycosylation status, and monoclonal antibody binding. Sources of likely conformational heterogeneity such as N-linked carbohydrates, flexible or mobile N and C termini, and variable internal loops were reduced or eliminated, and ligands such as CD4 and antigen-binding fragments (Fabs) of monoclonal antibodies were used to restrict conformational mobility as well as to alter the crystallization surface. Through successive cycles of manipulation involving 18 different variants, we succeeded in growing six different types of gp120 crystals. One of these, a ternary complex composed of gp120, its receptor CD4, and the Fab of the human neutralizing monoclonal antibody 17b, diffracts to a minimum Bragg spacing of at least 2.2 A and is suitable for structural analysis.

Mechanisms by which IkappaB proteins control NF-kappaB activity.

The biological activity of the transcription factor NF-kappaB is differentially controlled by three IkappaB proteins, Ikappa Balpha, Ikappa Bbeta, and Ikappa Bepsilon. We have examined the molecular basis for the differential inhibitory strengths of IkappaB proteins by constructing hybrid IkappaBs and found that the first ankyrin repeat of Ikappa Balpha is responsible for its strong inhibitory effect. Swapping a putative beta-turn within the first ankyrin repeat between the strong Ikappa Balpha and the weak IkappaBbeta inhibitors switches their in vivo inhibitory activity on NF-kappaB. The qualitatively distinct contacts made by this beta-turn in Ikappa Balpha and Ikappa Bbeta with NF-kappaB determine the efficiency by which IkappaBs sequester NF-kappaB to the cytoplasm, thus explaining their distinct effects on gene activity.

Crystal structure of mouse H2-M.

H2-M (HLA-DM in humans) resides in an acidic endosomal compartment, where it facilitates the loading of antigenic peptides into the peptide-binding groove of class II MHC. The crystal structure of a soluble form of H2-M has been solved to 3.1 A resolution, revealing a heterodimer with structural similarities to the MHC family of proteins. In contrast to its antigen-presenting cousins, the membrane distal alpha helices of H2-M pack closely together, occluding most of the binding groove except for a single large pocket near the center. The structure of H2-M has several unique features that may play a role in its function as a molecular chaperone and peptide exchange factor.

Novel Escherichia coli umuD' mutants: structure-function insights into SOS mutagenesis.

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD', the function of UmuD' has yet to be determined. In an attempt to elucidate the role of UmuD' in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD' plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD' mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD', three were nonsense mutations that resulted in a truncated UmuD' protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD'. All 17 umuD' mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD'.

Structure of a heparin-linked biologically active dimer of fibroblast growth factor.

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.