Di Xia

Crystal structure of the outer membrane active transporter FepA from Escherichia coli.

Integral outer membrane receptors for iron chelates and vitamin B 12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton–motive force of the inner membrane through physical interaction with TonB–ExbB–ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 Å resolution. Two distinct functional domains are revealed: (i) a 22–stranded β–barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N–terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB–mediated transport. The blockage of the pore suggests that the N–terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

Crystal structure of the receptor-binding domain of adenovirus type 5 fiberprotein at 1.7 Å resolution

BACKGROUND Adenoviral infection begins with the binding of virion to the surface of host cells. Specific attachment is achieved through interactions between host-cell receptors and the adenovirus fiber protein and is mediated by the globular carboxy-terminal domain of the adenovirus fiber protein, termed the carboxy-terminal knob domain. RESULTS The crystal structure of the carboxy-terminal knob domain of the adenovirus type 5 (Ad5) fiber protein has been determined at 1.7 A resolution. Each knob monomer forms an eight-stranded antiparallel beta-sandwich structure. In the crystal lattice, the knob monomers form closely interacting trimers which possess a deep surface depression centered around the three-fold molecular symmetry axis and three symmetry-related valleys. CONCLUSIONS The amino acid residues lining the wall of the central surface depression and the three symmetry-related floors of the valleys are strictly conserved in the knob domains of Ad5 and adenovirus type 2 (Ad2) fiber proteins, which share the same cellular receptor. The beta-sandwich structure of the knob monomer demonstrates a unique folding topology which is different from that of other known antiparallel beta-sandwich structures. The large buried surface area and numerous polar interactions in the trimer indicate that this form of the knob protein is predominant in solution, suggesting a possible assembly pathway for the native fiber protein.

Structure of the Receptor Binding Domain of Adenovirus Type 5 Fiber Protein

In this chapter we briefly describe functional and structural properties of the recombinant knob domain of the adenovirus type 5 (Ad5) fiber protein. We were able to demonstrate that this domain forms a trimer in solution and can compete with intact virus for binding to cellular receptors (Henry et al. 1994). We subsequently determined the crystal structure of knob at 1.7 A resolution (Xia et al. 1994). The knob monomers are β-sandwich structures and are arranged as closely associated trimers in the crystal. We compared sequences of knob domains from different adenovirus serotypes and found patterns of highly conserved and highly variable sequences on the knob surface. We interpreted the conserved regions as probable sites of interaction of the knob ligand with the cellular receptors and the variable regions as antigenic sites which define the structural differences among serotypes.

The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding

ATP‐binding cassette (ABC) transporters represent one of the largest families of proteins, and transport a variety of substrates ranging from ions to amphipathic anticancer drugs. The functional unit of an ABC transporter is comprised of two transmembrane domains and two cytoplasmic ABC ATPase domains. The energy of the binding and hydrolysis of ATP is used to transport the substrates across membranes. An ABC domain consists of conserved regions, the Walker A and B motifs, the signature (or C) region and the D, H and Q loops. We recently described the A‐loop (Aromatic residue interacting with the Adenine ring of ATP), a highly conserved aromatic residue ∼25 amino acids upstream of the Walker A motif that is essential for ATP‐binding. Here, we review the mutational analysis of this subdomain in human P‐glycoprotein as well as homology modeling, structural and data mining studies that provide evidence for a functional role of the A‐loop in ATP‐binding in most members of the superfamily of ABC transporters.

Inhibitor-complexed Structures of the Cytochrome bc1 from the Photosynthetic Bacterium Rhodobacter sphaeroides.

The cytochrome bc1 complex (bc1) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc1 complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc1), stabilized with the quinol oxidation (QP) site inhibitor stigmatellin alone or in combination with the quinone reduction (QN) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c1 subunit and a number of lipid and detergent molecules. Structural differences between Rsbc1 and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc1 complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.

A novel ATP‐dependent conformation in p97 N–D1 fragment revealed by crystal structures of disease‐related mutants

Mutations in p97, a major cytosolic AAA (ATPases associated with a variety of cellular activities) chaperone, cause inclusion body myopathy associated with Pagets disease of the bone and frontotemporal dementia (IBMPFD). IBMPFD mutants have single amino‐acid substitutions at the interface between the N‐terminal domain (N‐domain) and the adjacent AAA domain (D1), resulting in a reduced affinity for ADP. The structures of p97 N–D1 fragments bearing IBMPFD mutations adopt an atypical N‐domain conformation in the presence of Mg2+·ATPγS, which is reversible by ADP, showing for the first time the nucleotide‐dependent conformational change of the N‐domain. The transition from the ADP‐ to the ATPγS‐bound state is accompanied by a loop‐to‐helix conversion in the N–D1 linker and by an apparent re‐ordering in the N‐terminal region of p97. X‐ray scattering experiments suggest that wild‐type p97 subunits undergo a similar nucleotide‐dependent N‐domain conformational change. We propose that IBMPFD mutations alter the timing of the transition between nucleotide states by destabilizing the ADP‐bound form and consequently interfere with the interactions between the N‐domains and their substrates.

Crystallization and preliminary structure of beef heart mitochondrial cytochrome-bc1 complex

The method reported for isolation of ubiquinol-cytochrome-c reductase complex from submitochondrial particles was modified to yield a preparation for crystallization. The cytochrome bc1 complex was first crystallized in large thin plate form and diffracts X-rays to 7 A resolution in the presence of mother liquor. This crystalline complex was enzymatically active and contains ten protein subunits. It had 33 mol phospholipid and 0.6 mol ubiquinone per mol protein. With slightly modified crystallization conditions, different crystal forms were obtained. Crystals grown in the presence of 20% glycerol diffracted X-rays up to 2.9 A resolution using a synchrotron source. Four heavy atom derivatives have been obtained. The 3-D structure of the cytochrome bc1 complex was solved to 3.4 A resolution. Crystalline cytochrome bc1 complex is a dimer: most of the masses of core proteins I and II protrudes from the matrix side of the membrane, whereas the cytochrome b protein is located mainly within the membrane. There are 13 transmembrane helices in each monomer. Most of the mass of cytochrome c1 and iron-sulfur protein including their redox centers are located on the cytoplasmic side of the membrane. The distances between these redox centers have been determined, and several electron transfer inhibitor binding sites in the complex have been located.

Surface-modulated motion switch: capture and release of iron-sulfur protein in the cytochrome bc1 complex.

In the cytochrome bc1 complex, the swivel motion of the iron–sulfur protein (ISP) between two redox sites constitutes a key component of the mechanism that achieves the separation of the two electrons in a substrate molecule at the quinol oxidation (Qo) site. The question remaining is how the motion of ISP is controlled so that only one electron enters the thermodynamically favorable chain via ISP. An analysis of eight structures of mitochondrial bc1 with bound Qo site inhibitors revealed that the presence of inhibitors causes a bidirectional repositioning of the cd1 helix in the cytochrome b subunit. As the cd1 helix forms a major part of the ISP binding crater, any positional shift of this helix modulates the ability of cytochrome b to bind ISP. The analysis also suggests a mechanism for reversal of the ISP fixation when the shape complementarity is significantly reduced after a positional reorientation of the reaction product quinone. The importance of shape complementarity in this mechanism was confirmed by functional studies of bc1 mutants and by a structure determination of the bacterial form of bc1. A mechanism for the high fidelity of the bifurcated electron transfer is proposed.

Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli

Adhesion pili (fimbriae) play a critical role in initiating the events that lead to intestinal colonization and diarrheal disease by enterotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts an enormous global disease burden. We elucidate atomic structures of an ETEC major pilin subunit, CfaB, from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for 2 morphological forms of CFA/I fimbriae that are both observed in vivo: the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between these helical and extended forms. Our findings affirm the strong structural similarities seen between class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and class 1 pili (from bacteria that cause urinary, respiratory, and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein.

Structure of a multidrug transporter

Crystal structures of a mammalian multidrug efflux pump bound to peptide inhibitors may reveal drug-binding sites.