Structure of gramicidin D-RbCl complex at atomic resolution from low-temperature synchrotron data: interactions of double-stranded gramicidin channel contents and cations with channel wall.

Gramicidin D (gD) is a naturally occurring ionophoric antibiotic that forms membrane channels specific for monovalent cations. The crystal structure of the RbCl complex of gD has been determined at 1.14 A resolution from low-temperature (100 K) synchrotron-radiation data with a final R of 16%. The structure was refined with anisotropic temperature factors for all non-H atoms and with partial occupancies for many of them. The asymmetric unit in the crystal contains four crystallographically independent molecules that form two right-handed antiparallel double-stranded dimers. There are seven distinct rubidium-binding sites in each dimeric channel. The occupancy factors of Rb cations are between 0.11 and 0.35 and the total ion contents of the two crystallographically independent channels are 1.59 and 1.22 ions, respectively. Although each channel is 'chemically symmetrical', the side-chain conformations, the distributions of rubidium cations and their binding sites in the two independent channels are not. Cations are 'coordinated' by delocalized pi-electrons of three to five carbonyl groups that together with peptide backbone chains form the gramicidin channel walls. The water:cation ratio in the channel interior is four or five:one, and five or six waters separate Rb cations during their passage through the channel.

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide.

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

Phase changes in T(3)R(3)(f) human insulin: temperature or pressure induced?

The structure of T(3)R(3) hexameric human insulin has been determined at 100 K from two different crystals at 1.2 and 1.3 A resolution and refined to residuals of 0.169 and 0.176, respectively. Owing to a phase change, the c axis is double its room-temperature value and the asymmetric unit contains two independent TR(f) insulin dimers. Compared with the orientation in the room-temperature structure, one dimer undergoes a rotation about the c axis of -5 degrees, while the second is rotated +4 degrees. A superposition of the backbone atoms of the two independent dimers shows that the C(alpha) atoms of five residues within the R(f)-state monomers are displaced by more than 1.0 A; smaller displacements are observed for the T-state monomers. Four zinc ions lie on the crystallographic threefold axis and each forms bonds to three symmetry-related HisB10 N(varepsilon2) atoms from the T- and R(f)-state trimers. While three of the zinc ions are tetrahedrally coordinated with a chloride ion completing the coordination sphere, mixed tetrahedral/octahedral coordination is observed for one of the T-state zinc ions. The three symmetry-related "phenolic binding sites" in one hexamer contain water molecules and a glycerol molecule, but the same sites in the second hexamer are occupied by a zinc ion coordinated to an alternate conformation of HisB10, a symmetry-related HisB5 and two chloride ions. Two additional and partially occupied zinc ion sites are observed at the interface between the two independent dimers. One zinc ion is coordinated by a T-state HisB5 of one dimer, an R-state HisB5 of the second dimer and two water molecules; the second zinc ion is coordinated by an alternate side-chain conformation of the T-state HisB5 and three water molecules. The carboxyl group of one GluB13 side chain, which exists in two discrete conformations, appears to be protonated, because short contacts exist to a second carboxyl group or to a carbonyl O atom.

R6 hexameric insulin complexed with m-cresol or resorcinol.

The structures of three R(6) human insulin hexamers have been determined. Crystals of monoclinic m-cresol-insulin, monoclinic resorcinol-insulin and rhombohedral m-cresol-insulin diffracted to 1. 9, 1.9 and 1.78 A, respectively, and have been refined to residuals of 0.195, 0.179 and 0.200, respectively. In all three structures, a phenolic derivative is found to occupy the phenolic binding site, where it forms hydrogen bonds to the carbonyl O atom of CysA6 and the N atom of CysA11. Two additional phenolic derivative binding sites were identified within or between hexamers. The structures of all three hexamers are nearly identical, although a large displacement of the N-terminus of one B chain in both monoclinic structures results from coordination to a sodium ion which is located between symmetry-related hexamers. Other minor differences in structure arise from differences in packing in the monoclinic cell compared with the rhombohedral cell. Based upon the differences in conformation of the GluB13 side chains in T(6), T(3)R(f)(3) and R(6) hexamers, the deprotonation of these side chains appears to be associated with the T-->R conformational transition.

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities.

The three-dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4A resolution. The enzyme, which has both trypsin-like and chymotrypsin-like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin-like and chymotrypsin-like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities.

Gramicidin D conformation, dynamics and membrane ion transport.

The linear pentadecapeptide antibiotic, gramicidin D, a heterogeneous mixture of six components, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The conformation of gramicidin A in the solid state, in organic solvents, and in planar lipid bilayers and the relationship between the composition and the conformation of gramicidin and its selective transport of ions across membranes has been the subject of intense investigation for over 50 years. The x-ray crystal structure and nmr solution spectroscopy agree fully with one another and reveal that entirely different conformations of gramicidin are present in uncomplexed and ion complexed forms. Precise refinements of the three-dimensional structures of naturally occurring gramicidin D in crystals obtained from methanol, ethanol, and n-propanol demonstrate the unexpected presence of stable left-handed antiparallel double-helical heterodimers that vary with the crystallization solvent. The side chains of Trp residues in the three structures exhibit sequence-specific patterns of conformational preference. Tyr substitution for Trp at position 11 appears to favor beta ribbon formation and stabilization of the antiparallel double helix. This conformation acts as a template for gramicidin folding and nucleation of the different crystal forms. The fact that a minor component in a heterogeneous mixture influences aggregation and crystal nucleation has potential applications to other systems in which anomalous behavior is exhibited by aggregation of apparently homogeneous materials, such as the enigmatic behavior of prion proteins. The crystallographically determined structures of cesium, potassium, rubidium, and hydronium ion complexes of gramicidin A are in excellent agreement with the nmr structure determination of the cesium ion gramicidin complex in a methanol chloroform mixture (50 : 50). The right-handed antiparallel double stranded double helical structures (DSDHR) also exhibit geometric features compatible with the solid-state 15N and 2H nmr data recorded for gramicidin in planar lipid bilayers and attributed to the active form of gramicidin A. The DSDHR crystal structures reveal an ion channel with a single partially solvated cation distributed over three ion binding sites. The channel lumen is relatively smooth and electrostatically negative as required for cation passage, while the exterior is electrostatically neutral, a requirement for membrane insertion. The "coordination" of the Cs+ ion is achieved by interaction with the pi orbitals of the carbonyls which do not point toward the ions. The K+ binding sites, which are similar in position to Cs+ binding sites, are shifted off center slightly toward the wall of the channel.

Heterodimer formation and crystal nucleation of gramicidin D.

The linear pentadecapeptide antibiotic gramicidin D is a heterogeneous mixture of six components. Precise refinements of three-dimensional structures of naturally occurring gramicidin D in crystals obtained from methanol, ethanol, and n-propanol demonstrate the unexpected presence of stable left-handed antiparallel double-helical heterodimers that vary with the crystallization solvent. The side chains of Trp residues in the three structures exhibit sequence-specific patterns of conformational preference. Tyr substitution for Trp at position 11 appears to favor beta ribbon formation and stabilization of the antiparallel double helix that acts as a template for gramicidin folding and nucleation of different crystal forms. The fact that a minor component in a heterogeneous mixture influences aggregation and crystal nucleation has potential applications to other systems in which anomalous behavior is exhibited by aggregation of apparently homogeneous materials, such as the enigmatic behavior of prion proteins.

The conducting form of gramicidin A is a right-handed double-stranded double helix.

The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDH) reported here agree with 15N-NMR and CD data on the functional gramicidin D channel in lipid bilayers. These structures demonstrate single-file ion transfer through the channels. The results also indicate that previous crystal structure reports of a left-handed double-stranded double-helical dimer in complex with Cs+ and K+ salts may be in error and that our evidence points to the DSDH as the major conformer responsible for ion transport in membranes.

A thyrotropin-releasing hormone analogue: pGlu-Phe-D-Pro-psi [CN4]-NMe at 293 and 107 K.

Data have been measured at two temperatures, 293 K and 107 K, for a crystal of a thyrotropin-releasing hormone analogue, pGlu-Phe-D-Pro-psi [CN4]-NMe, C20H25N7O3, and the structures solved and refined. The tripeptide contains a tetrazole ring which mimics a cis-peptide bond at the C terminus. An intermolecular hydrogen bond exists between two molecules related by the twofold screw axis, resulting in infinite chains of hydrogen-bonded peptide molecules. Because of the folding and packing of the molecules, there are no intermolecular contacts of less than 4 A to the N atom of the phenylalanine residue.

Molecular interactions in binary solids: crystal structure of a cholesteryl ester solid solution.

The x-ray crystal structure analysis of a cholesteryl ester solid solution, cholesteryl undecanoate/cholesteryl laurate in a molar ratio 0.52/0.48, is described. The unit cell is monoclinic with a = 13.005(2) A, b = 9.005(1) A, c = 31.421(3) A, and beta = 90.82(1) degrees and the space group P2(1) with Z = 4 (two molecules per asymmetric unit). Thus, the d001 spacing is almost the value predicted by Vegard's law from the values for the pure compounds. The room-temperature crystal structure is very much like that of cholesteryl laurate monolayer I packing, in the form where the esterified fatty acid chains are fully extended, with no salient sign of conformational disorder seen in this binary solid. The final R factor for 6571 observed reflections is 0.097.

Molecular conformation and ion transport of cyclic and linear ionophores.

X-ray crystal structure determinations and energy-minimization techniques provide conformational data on the complexed and uncomplexed forms of ion transport antibiotics of the shuttle and channel types. In the solid state, hexadecaisoleucinomycin (HEXIL), an analogue of valinomycin, is observed as an asymmetric macrocycle stabilized by eight intramolecular (4----1) hydrogen bonds. The structure obtained from energy-minimization procedures exhibits a greater variation in phi and psi angles of chemically equivalent residues than does the crystallographically observed structure. The structure has eight carbonyl groups directed toward its interior and is capable of providing flexible coordination to a positively charged ion or molecule. These structural findings are consistent with the observed capacity of HEXIL to complex cesium ions, tetramethyl ammonium ions and acetylcholine. Gramicidin A is a pentadecapeptide that functions as a transmembrane channel for transporting monovalent cations. Uncomplexed gramicidin A crystallizes as a left-handed, antiparallel, double-stranded, helical dimer with 5.6 amino acid residues per turn. The helix has an overall length of 31 A and an average inner channel diameter of 4.8 A. The channel of this crystalline form does not contain ions or solvent molecules. Transporting ions through this channel could be achieved only by some expansion of the channel opening that would involve breaking and reforming hydrogen bonds that stabilize the double-stranded helix.

Polymorphic forms of 1,2-dipalmitoyl-sn-glycerol: a combined X-ray and electron diffraction study.

Quantitative crystallographic structure analyses are carried out for two polymorphic forms of 1,2-dipalmitoyl-sn-glycerol. A single crystal X-ray determination on the higher melting beta'L-form reveals that the hairpin conformer structure is essentially identical to that of the dilauroyl homolog reported earlier (I. Pascher, S. Sundell and H. Hauser (1981) J. Mol. Biol. 153, 791-806) with inclined acyl chain packing in the O perpendicular methylene subcell. Lamellar electron diffraction intensity data from epitaxially crystallized samples were used to determine the structure of the lower melting alpha L-form. The chains pack in the hexagonal subcell and are perpendicular to the lamellar surface. An appropriately oriented molecular model based on the beta'L-polymorph does not lead to a satisfactory structure solution but models based on the conformationally different 1,2-diglyceride moiety of several phospholipid structures does lead to a closer match to the observed diffraction data. In this proposed packing model for the alpha L-form, the hydroxyl oxygens are somewhat farther away from the unit cell origin than in the beta'L-form crystal structure, and, in combination with the different molecular conformation, this might explain the observed stability of this crystal polymorph against acyl shifts.

Epitaxial crystallization of alkane chain lipids for electron diffraction analysis.

Thin microcrystals of a wide variety of polymethylene chain materials, including n-alkanes, linear waxes, glycerides, a detergent, phospholipids and phospholipid analogs based on cyclopentane-1,2,3-triol, are epitaxially grown on naphthalene to give an orientation with long chain axes parallel to the best developed crystal face. These crystals, which represent a different orientation than those grown from solution, facilitate ab initio quantitative crystal structure analysis from electron diffraction intensity data from the projection yielding the most crystallographic information.

Studies on membrane fusion. II. Induction of fusion in pure phospholipid membranes by calcium ions and other divalent metals.

The effect of divalent metals on the interaction and mixing of membrane components in vesicles prepared from acidic phospholipids has been examined using freeze-fracture electron microscopy and differential scanning calorimetry. Ca2+, and to a certain extent Mg2+, induce extensive mixing of vesicle membrane components and drastic structural rearrangements to form new membranous structures. In contrast to the mixing of vesicle membrane components in the absence of Ca2+ described in the accompanying paper which occurs via diffusion of lipid molecules between vesicles, mixing of membrane components induced by Ca2+ or Mg2+ results from true fusion of entire vesicles. There appears to be a "threshold" concentration at which Ca2+ and Mg2+ become effective in inducing vesicle fusion and the threshold concentration varies for different acidic phospholipid species. Different phospholipids also vary markedly in their relative responsiveness to Ca2+ and Mg2+, with certain phospholipids being much more susceptible to fusion by Ca2+ than Mg2+. Vesicle fusion induced by divalent cations also requires that the lipids of the interacting membranes be in a "fluid" state (T greater than Tc). Fusion of vesicle membranes by Ca2+ and Mg2+ does not appear to be due to simple electrostatic charge neutralization. Rather the action of these cations in inducing fusion is related to their ability to induce isothermal phase transitions and phase separations in phospholipid membranes. It is suggested that under these conditions membranes become transiently susceptible to fusion as a result of changes in molecular packing and creation of new phase boundaries induced by Ca2+ (or Mg2+).

Lysolecithin and cholesterol interact stoichiometrically forming bimolecular lamellar structures in the presence of excess water, of lysolecithin or cholesterol.

The structural interaction of egg lysolecithin, derived from egg lecithin, and cholesterol in aqueous solution has been investigated using X-ray diffraction. When mixed in any proportions, either suspended in excess buffer or up to 85% lipid by dry weight, a separate lamellar phase containing equimolar proportions of lysolecithin and cholesterol forms, separate from excess water, or lysolecithin or cholesterol. The cholesterol disorders the crystalline chains of the lysolecithin. The equimolar phase is stable up to 50 degrees C unlike lysolecithin alone, which forms micelles, Thes results show that lysolecithin and cholesterol combine stoichiometrically in a stable complex. We propose as a structural model, that cholesterol fills the space of the missing fatty acyl chain making the lysolecithin more cylindrical rather than wedge shaped. This interaction could reduce both the lytic action of lysolecithin on membranes and its induction of cell fusion. It suggest another role of cholesterol in cell membranes: namely, to act as a stabilizer of bilayer structure by being a mobile component that can fill free volume in the hydrocarbon interior. Lysolecithin-cholesterol interaction may also be important in the early events of atherosclerosis where lysolecithin levels in vessel walls increase fivefold.

X-ray structure of racemic glycerol 1,2-(di-11-bromoundecanoate)-3-(-p-toluenesulfonate).

The single crystal x-ray structure of racemic glycerol 1,2-(di-11bromoundecanoate)-3-(p-toluenesulfonate), a sulfolipid analogous to the membrane phospholipids, reveals a folded conformation.