Convergent evolution in the supercoiling of prokaryotic flagellar filaments.

The supercoiling of bacterial and archaeal flagellar filaments is required for motility. Archaeal flagellar filaments have no homology to their bacterial counterparts and are instead homologs of bacterial type IV pili. How these prokaryotic flagellar filaments, each composed of thousands of copies of identical subunits, can form stable supercoils under torsional stress is a fascinating puzzle for which structural insights have been elusive. Advances in cryoelectron microscopy (cryo-EM) make it now possible to directly visualize the basis for supercoiling, and here, we show the atomic structures of supercoiled bacterial and archaeal flagellar filaments. For the bacterial flagellar filament, we identify 11 distinct protofilament conformations with three broad classes of inter-protomer interface. For the archaeal flagellar filament, 10 protofilaments form a supercoil geometry supported by 10 distinct conformations, with one inter-protomer discontinuity creating a seam inside of the curve. Our results suggest that convergent evolution has yielded stable superhelical geometries that enable microbial locomotion.

The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis.

Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of alternating ß-D-glucuronosyl and N-acetyl-α-D-glucosaminyl residues by exostosins. These enzymes generally possess two glycosyltransferase domains (GT47 and GT64)-each thought to add one type of monosaccharide unit to the backbone. Although previous structures of murine exostosin-like 2 (EXTL2) provide insight into the GT64 domain, the rest of the bi-domain architecture is yet to be characterised; hence, how the two domains co-operate is unknown. Here, we report the structure of human exostosin-like 3 (EXTL3) in apo and UDP-bound forms. We explain the ineffectiveness of EXTL3's GT47 domain to transfer ß-D-glucuronosyl units, and we observe that, in general, the bi-domain architecture would preclude a processive mechanism of backbone extension. We therefore propose that heparan sulfate backbone polymerisation occurs by a simple dissociative mechanism.

A duplicated fold is the structural basis for polynucleotide phosphorylase catalytic activity, processivity, and regulation.

BACKGROUND: Polynucleotide phosphorylase (PNPase) is a polyribonucleotide nucleotidyl transferase (E.C.2.7.7.8) that degrades mRNA in prokaryotes. Streptomyces antibioticus PNPase also assays as a guanosine 3'-diphosphate 5'-triphosphate (pppGpp) synthetase (E.C.2.7.6.5). It may function to coordinate changes in mRNA lifetimes with pppGpp levels during the Streptomyces lifecycle. RESULTS: The structure of S. antibioticus PNPase without bound RNA but with the phosphate analog tungstate bound at the PNPase catalytic sites was determined by X-ray crystallography and shows a trimeric multidomain protein with a central channel. The structural core has a novel duplicated architecture formed by association of two homologous domains. The tungstate derivative structure reveals the PNPase active site in the second of these core domains. Structure-based sequence analysis suggests that the pppGpp synthetase active site is located in the first core domain. CONCLUSIONS: This is the first structure of a PNPase and shows the structural basis for the trimer assembly, the arrangement of accessory RNA binding domains, and the likely catalytic residues of the PNPase active site. A possible function of the trimer channel is as a contribution to both the processivity of degradation and the regulation of PNPase action by RNA structural elements.

Crystal structure of the Escherichia coli RNA degradosome component enolase.

The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A. There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules, flexible loops close onto the active site. This closure mimics the conformation of the substrate-bound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a specific role to it, a structural role is possible.

A mutagenic study of the allosteric linkage of His(HC3)146 beta in haemoglobin.

We have examined the contribution of His(HC3)146 beta to the alkaline Bohr effect of human haemoglobin (HbA) by replacing it with Gln, using site-directed mutagenesis, and studying the structural and functional consequences. Oxygen equilibrium curves of the mutant show that the effect of pH on the oxygen affinity, the alkaline Bohr effect, is half that of HbA in the presence of chloride ion and less than 10% in its absence. Crystallographic analysis shows that the mutation introduced only small structural changes localized to the site of substitution, proving that the replacement of the hydrogen bond between the ionizable side-chain of His146 beta and Asp94 beta by a hydrogen bond between the unionizable side-chain of Gln146 beta and the same aspartate is solely responsible for the reduction of the alkaline Bohr effect. Our data confirm that His(HC3)146 beta is predominantly responsible for the chloride-independent component of the alkaline Bohr effect which results from the breaking of the hydrogen bond between His(HC3)146 beta and Asp(FG1)94 beta accompanying the transition from the quaternary deoxy to oxy-structure.

X-ray and solution studies of DNA oligomers and implications for the structural basis of A-tract-dependent curvature.

DNA containing short periodic stretches of adenine residues (known as A-tracts), which are aligned with the helical repeat, exhibit a pronounced macroscopic curvature. This property is thought to arise from the cumulative effects of a distinctive structure of the A-tract. It has also been observed by gel electrophoresis that macroscopic curvature is largely retained when inosine bases are introduced singly into A-tracts but decreases abruptly for pure I-tracts. The structural basis of this effect is unknown. Here we describe X-ray and gel electrophoretic analyses of several oligomers incorporating adenine or inosine bases or both. We find that macroscopic curvature is correlated with a distinctive base-stacking geometry characterized by propeller twisting of the base-pairs. Regions of alternating adenine and inosine bases display large propeller twisting comparable to that of pure A-tracts, whereas the values observed for pure I-tracts are significantly smaller. We also observe in the crystal structures that propeller twist leads to close cross-strand contacts between amino groups from adenine and cytosine bases, indicating an attractive NH-N interaction, which is analogous to the NH-O interaction proposed for A-tracts. This interaction also occurs between adenine bases across an A-T step and may explain in part the different behavior of A-T versus T-A steps in the context of A-tract-induced curvature. We also note that hydration patterns may contribute to propeller-twisted conformation. Based on the present data and other structural and biophysical studies, we propose that DNA macroscopic curvature is related to the structural invariance of A-tract and A-tract-like regions conferred by high propeller twist, cross-strand interactions and characteristic hydration. The implications of these findings to the mechanism of DNA bending are discussed.

Na+ transport via Na+/H+ antiport in Halobacterium halobium envelope vesicles.

Using H. halobium cell envelope vesicles containing either bacteriorhodopsin plus Na+ pump, bacteriorhodopsin alone, Na+ pump alone, or no light-responsive pigment altogether, it could be shown that the large majority of light energized Na+ extrusion in these mutants is linked to bacteriorhodopsin and to protonmotive force, and therefore must be facilitated by a Na+/H+ antiporter. Thus, the recently discovered primary Na+ pump makes only a minor contribution to light-mediated Na+ flux. The activity of the Na+/H+ antiporter appears to be independent of the presence of any photoreactive pigments, since an artifical electron donor will drive rapid Na+ extrusion in all of the vesicle preparations tested.

Crystal structure of the carbapenem intrinsic resistance protein CarG.

In the Gram-negative enterobacterium Erwinia (Pectobacterium) and Serratia sp. ATCC 39006, intrinsic resistance to the carbapenem antibiotic 1-carbapen-2-em-3-carboxylic acid is mediated by the CarF and CarG proteins, by an unknown mechanism. Here, we report a high-resolution crystal structure for the Serratia sp. ATCC 39006 carbapenem resistance protein CarG. This structure of CarG is the first in the carbapenem intrinsic resistance (CIR) family of resistance proteins from carbapenem-producing bacteria. The crystal structure shows the protein to form a homodimer, in agreement with results from analytical gel filtration. The structure of CarG does not show homology with any known antibiotic resistance proteins nor does it belong to any well-characterised protein structural family. However, it is a close structural homologue of the bacterial inhibitor of invertebrate lysozyme, PliI-Ah, with some interesting structural variations, including the absence of the catalytic site responsible for lysozyme inhibition. Both proteins show a unique ß-sandwich fold with short terminal α-helices. The core of the protein is formed by stacked anti-parallel sheets that are individually very similar in the two proteins but differ in their packing interface, causing the splaying of the two sheets in CarG. Furthermore, a conserved cation binding site identified in CarG is absent from the homologue.

A "mechanistic" explanation of the multiple helical forms adopted by bacterial flagellar filaments.

The corkscrew-like flagellar filaments emerging from the surface of bacteria such as Salmonella typhimurium propel the cells toward nutrient and away from repellents. This kind of motility depends upon the ability of the flagellar filaments to adopt a range of distinct helical forms. A filament is typically constructed from ~30,000 identical flagellin molecules, which self-assemble into a tubular structure containing 11 near-longitudinal protofilaments. A "mechanical" model, in which the flagellin building block has the capacity to switch between two principal interfacial states, predicts that the filament can assemble into a "canonical" family of 12 distinct helical forms, each having unique curvature and twist: these include two "extreme" straight forms having left- and right-handed twists, respectively, and 10 intermediate helical forms. Measured shapes of the filaments correspond well with predictions of the model. This report is concerned with two unanswered questions. First, what properties of the flagellin determine which of the 12 discrete forms is preferred? Second, how does the interfacial "switch" work, at a molecular level? Our proposed solution of these problems is based mainly on a detailed examination of differences between the available electron cryo-microscopy structures of the straight L and R filaments, respectively.

Geometric principles in the assembly of α-helical bundles.

α-Helical coiled coils are usually stabilized by hydrophobic interfaces between the two constituent α-helices, in the form of 'knobs-into-holes' packing of non-polar residues arranged in repeating heptad patterns. Here we examine the corresponding 'hydrophobic cores' that stabilize bundles of four α-helices. In particular, we study three different kinds of bundle, involving four α-helices of identical sequence: two pack in a parallel and one in an anti-parallel orientation. We point out that the simplest way of understanding the packing of these 4-helix bundles is to use Crick's original idea that the helices are held together by 'hydrophobic stripes', which are readily visualized on the cylindrical surface lattice of the α-helices; and that the 'helix-crossing angle'--which determines, in particular, whether supercoiling is left- or right-handed--is fixed by the slope of the lattice lines that contain the hydrophobic residues. In our three examples the constituent α-helices have hydrophobic repeat patterns of 7, 11 and 4 residues, respectively; and we associate the different overall conformations with 'knobs-into-holes' packing along the 7-, 11- and 4-start lines, respectively, of the cylindrical surface lattices of the constituent α-helices. For the first two examples, all four interfaces between adjacent helices are geometrically equivalent; but in the third, one of the four interfaces differs significantly from the others. We provide a geometrical explanation for this non-equivalence in terms of two different but equivalent ways of assembling this bundle, which may possibly constitute a bistable molecular 'switch' with a coaxial throw of about 12 Å. The geometrical ideas that we deploy in this paper provide the simplest and clearest description of the structure of helical bundles. In an appendix, we describe briefly a computer program that we have devised in order to search for 'knobs-into-holes' packing between α-helices in proteins.

Amyloidogenicity and aggregate cytotoxicity of human glucagon-like peptide-1 (hGLP-1).

The potential of human glucagon-like peptide-1 (hGLP-1) as a therapeutic agent is limited by its high aggregation propensity. We show that hGLP-1 forms amyloid-like structures that are preceded by cytotoxic aggregates, suggesting that aggregation of biopharmaceuticals could present a cytotoxic risk to patients besides the reported increased risk in immunogenicity.

Complementing structural information of modular proteins with small angle neutron scattering and contrast variation.

Many macromolecules in the cell function by forming multi-component assemblies. We have applied the technique of small angle neutron scattering to study a nucleic acid-protein complex and a multi-protein complex. The results illustrate the versatility and applicability of the method to study macromolecular assemblies. The neutron scattering experiments, complementing X-ray solution scattering data, reveal that the conserved catalytic domain of RNase E, an essential ribonuclease in Escherichia coli (E. coli), undergoes a marked conformational change upon binding a 5'monophosphate-RNA substrate analogue. This provides the first evidence in support of an allosteric mechanism that brings about RNA substrate cleavage. Neutron contrast variation of the multi-protein TIM10 complex, a mitochondrial chaperone assembly comprising the subunits Tim9 and Tim10, has been used to determine a low-resolution shape reconstruction of the complex, highlighting the integral subunit organization. It shows characteristic features involving protrusions that could be assigned to the six subunits forming the complex.

Structure, mechanism and catalytic duality of thiamine-dependent enzymes.

Thiamine is an essential cofactor that is required for processes of general metabolism amongst all organisms, and it is likely to have played a role in the earliest stages of the evolution of life. Here, we review from a structural perspective the enzymatic mechanisms that involve this cofactor. We explore asymmetry within homodimeric thiamine diphosphate (ThDP)-dependent enzyme structures and discuss how this may be correlated with the kinetic properties of half-of-the-sites reactivity, and negative cooperativity. It is likely these structural and kinetic hallmarks may arise through reciprocal coupling of active sites. This mode of communication between distant active sites is not unique to ThDP-dependent enzymes, but is widespread in other classes of oligomeric enzyme. Thus, it appears likely to be a general phenomenon reflecting a powerful mechanism of accelerating the rate of a chemical pathway. Finally, we speculate on the early evolutionary history of the cofactor and its ancient association with protein and RNA.

Design and chance in the self-assembly of macromolecules.

The principles of self-assembly are described for naturally occurring macromolecules and for complex assemblies formed from simple synthetic constituents. Many biological molecules owe their function and specificity to their three-dimensional folds, and, in many cases, these folds are specified entirely by the sequence of the constituent amino acids or nucleic acids, and without the requirement for additional machinery to guide the formation of the structure. Thus sequence may often be sufficient to guide the assembly process, starting from denatured components having little or no folds, to the completion state with the stable, equilibrium fold that encompasses functional activity. Self-assembly of homopolymeric structures does not necessarily preserve symmetry, and some polymeric assemblies are organized so that their chemically identical subunits pack stably in geometrically non-equivalent ways. Self-assembly can also involve scaffolds that lack structure, as seen in the multi-enzyme assembly, the degradosome. The stable self-assembly of lipids into dynamic membraneous sheets is also described, and an example is shown in which a synthetic detergent can assemble into membrane layers.

Crystallographic analysis of mutant human haemoglobins made in Escherichia coli.

The expression of beta-globin in Escherichia coli has enabled us to study the functional role of individual amino-acid residues in haemoglobin (Hb) by site-directed mutagenesis. In contrast to mammalian Hbs, some teleost fish haemoglobins show a drastic lowering of oxygen affinity and cooperativity at low pH, a phenomenon known as the Root effect. We have produced the two mutant haemoglobins Hb Nymphéas [Cys(F9)93 beta----Ser] and Hb Daphne [His(H21)143 beta----Arg, Cys(F9)93 beta----Ser] to investigate this allosteric property. Although these substitutions were thought to be responsible for the Root effect, Hb Nymphéas and Hb Daphne show an increased oxygen affinity and a reduced effect of pH on oxygen affinity. Our X-ray crystallographic studies show that the hydroxyl group of Ser 93 beta forms a hydrogen bond with Asp 94 beta which is in equilibrium with the salt bridge between Asp 94 beta and His 146 beta. The oxygen-binding properties of Hbs Nymphéas and Daphne are accounted for by the partial disruption of the salt bridge.

On the mechanism of DNA binding by nuclear hormone receptors: a structural and functional perspective.

The nuclear hormone receptor DNA-binding domain consists of two zinc finger-like modules whose amino acids are highly conserved among the members of the receptor superfamily. In this review, we describe the various genetic, biochemical, and structural experiments that have been carried out primarily for the DNA-binding domains of the glucocorticoid and estrogen receptors. We describe how the structural and functional information have permitted us to predict properties of the DNA-binding domains of other nuclear receptors. We postulate how receptors discriminate closely related response elements through sequence-specific contacts and distinguish symmetry of target sites through protein-protein interactions. This mechanism explains in part how the receptors regulate diverse sets of genes from a limited repertoire of core response elements. Lastly, we describe the stereochemical basis of nuclear receptor dysfunction in certain clinical disorders.

X-ray crystallographic and functional studies of human haemoglobin mutants produced in Escherichia coli.

Human beta-globin was produced in Escherichia coli as a cleavable fusion protein using the expression vector pLcII [Nagai and Thøgersen, Nature 301, p. 810, 1984]. The fusion protein CIIFX beta-globin was purified under denaturing conditions to homogeneity and the authentic beta-globin was liberated by blood coagulation factor Xa. beta-Globin was then folded and reconstituted with haem and alpha-subunits to form fully functional alpha 2 beta 2-tetramers [Nagai et al., Proc. natn. Acad. Sci. USA 82, p. 7252, 1985]. This has enabled us to produce mutants with amino acid substitutions in the beta-subunit at will and in sufficient quantities to study their oxygen-binding properties and three-dimensional structures. We have crystallised three mutants, Hb Nympheas [Cys-93 beta----Ser], Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg] and Hb Sandra [Cys-93 beta----Ser, Asp-94 beta----Glu], and have solved their structures to high resolution by x-ray crystallography.

The structure of an oligo(dA).oligo(dT) tract and its biological implications.

Poly(dA).poly(dT) has unusual properties in that it cannot associate into nucleosomes and short, phased runs of it cause DNA bending. The crystal structure of a B-type DNA dodecamer containing a homopolymeric run of six A.T base pairs shows that this region possesses special structural features, including a system of bifurcated hydrogen bonds, which explains some of the properties of this simple homopolymer.

DNA target selectivity by the vitamin D3 receptor: mechanism of dimer binding to an asymmetric repeat element.

The 1,25-dihydroxyvitamin D3 receptor, like other members of the nuclear receptor superfamily, forms dimers in solution that are probably stabilized by a dyad symmetrical interface formed by the ligand-binding domain. This receptor, however, recognizes DNA targets that are not dyad symmetric but rather are organized as direct repeats of a hexameric sequence with a characteristic 3-bp spacing. Using molecular modeling and site-directed mutagenesis, we have identified regions within the vitamin D3 receptor zinc finger region that confer selectivity for direct repeats with appropriate spacing. Reflecting the organization of the DNA target, these regions, mapping to the tip of the first zinc finger module and the N and C termini of the second finger module, direct asymmetrical protein-protein contacts. A stereochemical model is proposed for these interactions.