Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Experimental evidence for proton transfer via a hydrogen-bonded network in a membrane protein is presented. Bacteriorhodopsin's proton transfer mechanism on the proton uptake pathway between Asp-96 and the Schiff base in the M-to-N transition was determined. The slowdown of this transfer by removal of the proton donor in the Asp-96-->Asn mutant can be accelerated again by addition of small weak acid anions such as azide. Fourier-transform infrared experiments show in the Asp-96-->Asn mutant a transient protonation of azide bound to the protein in the M-to-N transition and, due to the addition of azide, restoration of the IR continuum band changes as seen in wild-type bR during proton pumping. The continuum band changes indicate fast proton transfer on the uptake pathway in a hydrogen-bonded network for wild-type bR and the Asp-96-->Asn mutant with azide. Since azide is able to catalyze proton transfer steps also in several kinetically defective bR mutants and in other membrane proteins, our finding might point to a general element of proton transfer mechanisms in proteins.

On the electronic nature of low-barrier hydrogen bonds in enzymatic reactions

The electronic nature of low-barrier hydrogen bonds (LBHBs) in enzymatic reactions is discussed based on combined low temperature neutron and x-ray diffraction experiments and on high level ab initio calculations by using the model substrate benzoylacetone. This molecule has a LBHB, as the intramolecular hydrogen bond is described by a double-well potential with a small barrier for hydrogen transfer. From an “atoms in molecules” analysis of the electron density, it is found that the hydrogen atom is stabilized by covalent bonds to both oxygens. Large atomic partial charges on the hydrogen-bonded atoms are found experimentally and theoretically. Therefore, the hydrogen bond gains stabilization from both covalency and from the normal electrostatic interactions found for long, weak hydrogen bonds. Based on comparisons with other systems having short-strong hydrogen bonds or LBHBs, it is proposed that all short-strong and LBHB systems possess similar electronic features of the hydrogen-bonded region, namely polar covalent bonds between the hydrogen atom and both heteroatoms in question.

A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment

The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-β-d-maltoside and cyclohexyl-hexyl-β-d-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 Å (1 Å = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

Propagating structure of Alzheimer’s β-amyloid(10–35) is parallel β-sheet with residues in exact register

The pathognomonic plaques of Alzheimer’s disease are composed primarily of the 39- to 43-aa β-amyloid (Aβ) peptide. Crosslinking of Aβ peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Aβ. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Aβ peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Aβ. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 Å. Analysis of these data establish that this central core of Aβ consists of a parallel β-sheet structure in which identical residues on adjacent chains are aligned directly, i.e., in register. Our data, in conjunction with existing structural data, establish that the Aβ fibril is a hydrogen-bonded, parallel β-sheet defining the long axis of the Aβ fibril propagation.

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s−1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.

Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains

The 2.8-Å crystal structure of the complex formed by estradiol and the human estrogen receptor-α ligand binding domain (hERαLBD) is described and compared with the recently reported structure of the progesterone complex of the human progesterone receptor ligand binding domain, as well as with similar structures of steroid/nuclear receptor LBDs solved elsewhere. The hormone-bound hERαLBD forms a distinctly different and probably more physiologically important dimer interface than its progesterone counterpart. A comparison of the specificity determinants of hormone binding reveals a common structural theme of mutually supported van der Waals and hydrogen-bonded interactions involving highly conserved residues. The previously suggested mechanism by which the estrogen receptor distinguishes estradiol’s unique 3-hydroxy group from the 3-keto function of most other steroids is now described in atomic detail. Mapping of mutagenesis results points to a coactivator-binding surface that includes the region around the “signature sequence” as well as helix 12, where the ligand-dependent conformation of the activation function 2 core is similar in all previously solved steroid/nuclear receptor LBDs. A peculiar crystal packing event displaces helix 12 in the hERαLBD reported here, suggesting a higher degree of dynamic variability than expected for this critical substructure.

Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-Å resolution

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-Å resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an ɛ-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.

Reversible encapsulation by self-assembling resorcinarene subunits

Encapsulation complexes are assemblies in which a reversibly formed host more or less completely surrounds guest molecules. Host structures held together by hydrogen bonds have lifetimes in organic solvents of milliseconds to hours, long enough to directly observe the encapsulated guest by NMR spectroscopy. We describe here the action of alkyl ammonium compounds as guests that gather up to six molecules of the host module to form encapsulation complexes. The stoichiometry of the complexes—the largest hydrogen-bonded host capsules to date—is determined by the size and concentration of the guest.

Is water structure around hydrophobic groups clathrate-like?

The term "clathrate structure" is quantified for solvation of nonpolar groups by enumerating hydrogen-bonded ring sizes both in the solvation shell and through the shell-bulk interface and comparing it to a bulk control using the ST4 water model. For clathrate-like structure to be evident, the distributions along the hydrophobic surface are expected to be dominated by pentagons, with significant depletion of hexagons and larger polygons. While the distribution in this region is indeed distinguished by a large number of pentagons, there are significant contributions from hexagons and larger rings as well. Calculated polygon distributions through the shell-bulk interface indicate that when water structure is highly cooperative along the hydrophobic surface, hydrogen-bonded pathways leading back into bulk are then reduced. These results are qualitatively consistent with the observation that hydrophobicity is proportional to the nonpolar solute surface area.