Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin.

The cytoskeletal proteins talin and vinculin are localized at cell-matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F-actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four- and five-helix bundles that harbor amphipathic α-helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four-helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α-helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four-helix bundle.

The role of the TolC family in protein transport and multidrug efflux. From stereochemical certainty to mechanistic hypothesis.

Gram-negative bacteria are enveloped by a system of two membranes, and they use specialized multicomponent, energy-driven pumps to transport molecules directly across this double-layered partition from the cell interior to the extra-cellular environment. One component of these pumps is embedded in the outer-membrane, and the paradigm for its structure and function is the TolC protein from Escherichia coli. A common component of a wide variety of efflux pumps, TolC and its homologues are involved in the export of chemically diverse molecules ranging from large protein toxins, such as alpha-hemolysin, to small toxic compounds, such as antibiotics. TolC family members thus play important roles in conferring pathogenic bacteria with both virulence and multidrug resistance. These pumps assemble reversibly in a transient process that brings together TolC or its homologue, an inner-membrane-associated periplasmic component, an integral inner-membrane translocase and the substrate itself. TolC can associate in this fashion with a variety of different partners to participate in the transport of diverse substrates. We review here the structure and function of TolC and the other components of the efflux/transport pump.

Molecular shapes of transcription factors TFIIB and VP16 in solution: implications for recognition.

The molecular shapes of transcription factors TFIIB and VP16 have been studied by small-angle X-ray scattering (SAXS). We interpret the shapes and discuss the implications for the specific recruitment of these proteins into regulatory assemblies. Human transcription factor TFIIB, a universal component of the transcription preinitiation complex, has a triangular form resulting from intramolecular associations between its two principal structural domains. A segment linking the two domains appears to be conformationally flexible. The solution shape of TFIIB can be well fitted with the crystal structure of the DNA-bound C-terminal domain together with the NMR structure of the N-terminal domain; however, the shape cannot accommodate the NMR structure of the isolated C-terminal domain. We discuss how the conformational differences between the solution structures of the isolated C-terminal domain and the intact protein might result from interdomain allostery. Docking the SAXS shape of intact TFIIB into the preinitiation complex suggests that the flexible linker region may contact the 3' flanking region of the TATA element in the major groove. Transcription rates can be enhanced by activator proteins, and the classical example is the herpes simplex virus factor VP16 (alpha-TIF), which associates with cellular transcription factors, including TFIIB. The shape reconstruction of VP16 from its SAXS profile reveals a globular structural core that can be well modeled by the crystal structure of a conserved, central region of the protein. However, the carboxy terminus extends from this core and is essentially disordered. As it makes defined protein-protein interactions in the activation complex, the flexible segment is likely to condense upon assembly with its partners.

How to untwist an alpha-helix: structural principles of an alpha-helical barrel.

Recent crystallographic studies have revealed that 12 alpha-helices can pack in an anti-parallel fashion to form a hollow cylinder of nearly uniform radius. In this architecture, which we refer to as an alpha-barrel, the helices are inclined with respect to the cylindrical axis, and thus they curve and twist. As with conventional coiled-coils, the helices of the barrel associate via "knobs-into-holes" interactions; however, their packing is distinct in several important ways. First, the alpha-barrel helices untwist in comparison with the helices found in two-stranded coiled-coils and, as a consequence of this distortion, their knobs approach closely one end of the complementary holes. This effect defines a requirement for particular size and shape of the protruding residues, and it is associated with a relative axial translation of the paired helices. Second, as each helix packs laterally with two neighbours, the helices have two sequence patterns that are phased to match the two interfaces. The two types of interface are not equivalent and, as one travels around the circumference of the cylinder's interior, they alternate between one type where the knobs approach the holes straight-on, and a second type in which they are inclined. The choice of amino acid depends on the interface type, with small hydrophobic side-chains preferred for the direct contacts and larger aliphatic side-chains for the inclined contacts. Third, small residues are found preferentially on the inside of the tube, in order to make the "wedge" angle between helices compatible with a 12-member tube. Finally, hydrogen-bonding interactions of side-chains within and between helices support the assembly. Using these salient structural features, we present a sequence template that is compatible with some underlying rules for the packing of helices in the barrel, and which may have application to the design of higher-order assemblies from peptides, such as nano-tubes. We discuss the general implications of relative axial translation in coiled-coils and, in particular, the potential role that this movement could play in allosteric mechanisms.

Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export.

Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-A crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit--a 'channel-tunnel' over 140 A long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein-protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.

Oxidation of selenomethionine: some MADness in the method!

Since it was first reported, the multiwavelength anomalous diffraction (MAD) technique for the determination of protein structures has become widely accepted and increasingly popular. Here, it is demonstrated that the anomalous signal from selenomethione (SeMet) substituted proteins can be significantly enhanced by oxidation.

Refined structures of two insertion/deletion mutants probe function of the maltodextrin binding protein.

The X-ray structures of the maltose bound forms of two insertion/deletion mutants of the Escherichia coli maltodextrin binding protein, MalE322 and MalE178, have been determined and refined. MalE322 involves a one residue deletion, two residue insertion in a hinge segment connecting the two (N and C) domains of the protein, an area already identified as being critical for the correct functioning of the protein. MalE178 involves a nine residue deletion and two residue insertion in a helix at the periphery of the C-domain. The function of both mutant proteins is similar to the wild-type, although MalE322 increases the ability to transport maltose and maltodextrin whilst inhibiting the ability of the cell to grow on dextrins. Both proteins exhibit very localized and conservative conformational changes due to their mutations. The structure of MalE322 shows some deformation of the third hinge strand, indicating the likely cause of change in its biochemistry. MalE178 is stable and its activity virtually unchanged from the wild-type. This is most likely due to the long distance of the mutation from the binding site and conservation of the number of interactions between the area around the deletion site and the main body of the protein.

Refined 1.8-A structure reveals the mode of binding of beta-cyclodextrin to the maltodextrin binding protein.

The maltodextrin binding protein from Escherichia coli serves as the initial receptor for both the active transport of and chemotaxis toward a range of linear maltose sugars. The X-ray structures of both the maltose-bound and sugar-free forms of the protein have been previously described [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219; Sharff, A. J., Rodseth, L. E., Spurlino, J. C., & Quocho, F. A. (1992) Biochemistry 31, 10657-10663]. The X-ray crystal structure of the maltodextrin binding protein complexed with cyclomaltoheptaose (beta-cyclodextrin) has been determined from a single crystal. The structure has been refined to a final R-value of 21% at 1.8-A resolution. Although not a physiological ligand for the maltodextrin binding protein, beta-cyclodextrin has been shown to bind with a Kd of the same order as those of the linear maltodextrin substrates. The observed structure shows that the complexed protein remains in the fully open conformation and is almost identical to the structure of the unliganded protein. The sugar sits in the open cleft with three glucosyl units bound to the C-domain at the base of the cleft, in a similar position to maltotriose, the most tightly bound ligand. The top of the ring is loosely bound to the upper edge of the cleft on the N-domain. The sugar makes a total of 94 productive interactions (of less than 4.0-A length) with the protein and with bound water molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis.

The periplasmic maltodextrin binding protein of Escherichia coli serves as an initial receptor for the active transport of and chemotaxis toward maltooligosaccharides. The three-dimensional structure of the binding protein complexed with maltose has been previously reported [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219]. Here we report the structure of the unliganded form of the binding protein refined to 1.8-A resolution. This structure, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein. The unliganded structure shows a rigid-body "hinge-bending" between the two globular domains by approximately 35 degrees, relative to the maltose-bound structure, opening the sugar binding site groove located between the two domains. In addition, there is an 8 degrees twist of one domain relative to the other domain. The conformational changes observed between this structure and the maltose-bound structure are consistent with current models of maltose/maltodextrin transport and maltose chemotaxis and solidify a mechanism for receptor differentiation between the ligand-free and ligand-bound forms in signal transduction.

Refined 2.5 A structure of murine adenosine deaminase at pH 6.0.

The X-ray structure of murine adenosine deaminase complexed with the transition-state analogue 6-hydroxyl-1,6-dihydropurine ribonucleoside has been determined from a single crystal grown at pH 4.2 and transferred to mother liquor of increasing pH up to a final pH of 6.0 prior to data collection. The structure has been refined to 2.5 A to a final crystallographic R-factor of 20% using phases from the previously refined 2.4 A structure at pH 4.2. Kinetic measurements show that the enzyme is only 20% active at pH 4.2 whereas it is fully active between pH 6.0 and pH 8.5. The refined structures at either pH are essentially the same. Consideration of the pKa values of the key catalytic residues and the mechanism proposed on the basis of the structure suggests that the ionization state of these residues is largely responsible for the pH dependence on activity.