Zinc-dependent dimers observed in crystals of human endostatin

The crystal structure of human endostatin reveals a zinc-binding site. Atomic absorption spectroscopy indicates that zinc is a constituent of both human and murine endostatin in solution. The human endostatin zinc site is formed by three histidines at the N terminus, residues 1, 3, and, 11, and an aspartic acid at residue 76. The N-terminal loop ordered around the zinc makes a dimeric contact in human endostatin crystals. The location of the zinc site at the amino terminus, immediately adjacent to the precursor cleavage site, suggests the possibility that the zinc may be involved in activation of the antiangiogenic activity following cleavage from the inactive collagen XVIII precursor or in the cleavage process itself.

A designed beta-hairpin peptide in crystals.

Beta-hairpin structures have been crystallographically characterized only in very short acyclic peptides, in contrast to helices. The structure of the designed beta-hairpin, t-butoxycarbonyl-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe in crystals is described. The two independent molecules of the octapeptide fold into almost ideal beta-hairpin conformations with the central D-Pro-Gly segment adopting a Type II' beta-turn conformation. The definitive characterization of a beta-hairpin has implications for de novo peptide and protein design, particularly for the development of three- and four-stranded beta-sheets.

Organic protomolecule assembly in igneous minerals

C—H stretching bands, νCH, in the infrared spectrum of single crystals of nominally high purity, of laboratory-grown MgO, and of natural upper mantle olivine, provide an “organic” signature that closely resembles the symmetrical and asymmetrical C—H stretching modes of aliphatic —CH2 units. The νCH bands indicate that H2O and CO2, dissolved in the matrix of these minerals, converted to form H2 and chemically reduced C, which in turn formed C—H entities, probably through segregation into defects such as dislocations. Heating causes the C—H bonds to pyrolyze and the νCH bands to disappear, but annealing at 70°C causes them to reappear within a few days or weeks. Modeling dislocations in MgO suggests that the segregation of C can lead to Cx chains, x = 4, with the terminal C atoms anchored to the MgO matrix by bonding to two O−. Allowing H2 to react with such Cx chains leads to [O2C(CH2)2CO2] or similar precipitates. It is suggested that such Cx—Hy—Oz entities represent protomolecules from which derive the short-chain carboxylic and dicarboxylic and the medium-chain fatty acids that have been solvent-extracted from crushed MgO and olivine single crystals, respectively. Thus, it appears that the hard, dense matrix of igneous minerals represents a medium in which protomolecular units can be assembled. During weathering of rocks, the protomolecular units turn into complex organic molecules. These processes may have provided stereochemically constrained organics to the early Earth that were crucial to the emergence of life.

Comparing protein–ligand interactions in solution and single crystals by Raman spectroscopy

By using a Raman microscope, we show that it is possible to probe the conformational states in protein crystals and crystal fragments under growth conditions (in hanging drops). The flavin cofactor in the enzyme para-hydroxybenzoate hydroxylase can assume two conformations: buried in the protein matrix (“in”) or essentially solvent-exposed (“out”). By using Raman difference spectroscopy, we previously have identified characteristic flavin marker bands for the in and out conformers in the solution phase. Now we show that the flavin Raman bands can be used to probe these conformational states in crystals, permitting a comparison between solution and crystal environments. The in or out marker bands are similar for the respective conformers in the crystal and in solution; however, significant differences do exist, showing that the environments for the flavin's isoalloxazine ring are not identical in the two phases. Moreover, the Raman-band widths of the flavin modes are narrower for both in and out conformers in the crystals, indicating that the flavin exists in a more limited range of closely related conformational states in the crystal than in solution. In general, the ability to compare detailed Raman data for complexes in crystals and solution provides a means of bridging crystallographic and solution studies.

Electron tunneling in protein crystals

The current understanding of electron tunneling through proteins has come from work on systems where donors and acceptors are held at fixed distances and orientations. The factors that control electron flow between proteins are less well understood, owing to uncertainties in the relative orientations and structures of the reactants during the very short time that tunneling occurs. As we report here, the way around such structural ambiguity is to examine oxidation–reduction reactions in protein crystals. Accordingly, we have measured and analyzed the kinetics of electron transfer between native and Zn-substituted tuna cytochrome c (cyt c) molecules in crystals of known structure. Electron transfer rates [(320 s−1 for *Zn-cyt c → Fe(III)-cyt c; 2000 s−1 for Fe(II)-cyt c → Zn-cyt c+)] over a Zn–Fe distance of 24.1 Å closely match those for intraprotein electron tunneling over similar donor–acceptor separations. Our results indicate that van der Waals interactions and water-mediated hydrogen bonds are effective coupling elements for tunneling across a protein–protein interface.

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52–288). The structure resolved to 2.8 Å is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 Å apart. A 26-aa α-helix, α6, links the C-terminal domain to the catalytic core. A kink in one of the two α6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein–DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52–210), independently determined at 1.6-Å resolution, is identical to the core domain within the two-domain 52–288 structure.

Peptide hybrids containing α- and β-amino acids: Structure of a decapeptide β-hairpin with two facing β-phenylalanine residues

A β-hairpin conformation has been characterized in crystals of the decapeptide t-butoxycarbonyl-Leu-Val-βPhe-Val-DPro-Gly-Leu-βPhe-Val-Val-methyl ester [βPhe; (S)-β3 homophenylalanine] by x-ray diffraction. The polypeptide chain reversal is nucleated by the centrally positioned DPro-Gly segment, which adopts a type-I′ β-turn conformation. Four intramolecular cross-strand hydrogen bonds stabilize the peptide fold. The βPhe(3) and βPhe(8) residues occupy facing positions on the hairpin, with the side chains projecting on opposite faces of the β-sheet. At the site of insertion of β-residues, the polarity of the peptide units along each strand reverses, as compared with the α-peptide segments. In this analog, a small segment of a polar sheet is observed, where adjacent CO and NH groups line up in opposite directions in each strand. In the crystal, an extended β-sheet is formed by hydrogen bonding between strands of antiparallel pairs of β-hairpins. The crystallographic parameters for C65H102N10O13⋅ 3H2O are: space group P212121; a = 19.059(8) Å, b = 19.470(2) Å, c = 21.077(2) Å; Z = 4; agreement factor R1 = 9.12% for 3,984 data observed >4σ(F) and a resolution of 0.90 Å.

The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin.

In the previously determined structure of mitochondrial F1-ATPase determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. AMP-PNP and ADP are bound to subunits beta TP and beta DP, respectively, and the third beta-subunit (beta E) has no bound nucleotide. The efrapeptins are a closely related family of modified linear peptides containing 15 amino acids that inhibit both ATP synthesis and hydrolysis by binding to the F1 catalytic domain of F1F0-ATP synthase. In crystals of F1-ATPase grown in the presence of both nucleotides and inhibitor, efrapeptin is bound to a unique site in the central cavity of the enzyme. Its binding is associated with small structural changes in side chains of F1-ATPase around the binding pocket. Efrapeptin makes hydrophobic contacts with the alpha-helical structure in the gamma-subunit, which traverses the cavity, and with subunit beta E and the two adjacent alpha-subunits. Two intermolecular hydrogen bonds could also form. Intramolecular hydrogen bonds probably help to stabilize efrapeptin's two domains (residues 1-6 and 9-15, respectively), which are connected by a flexible region (beta Ala-7 and Gly-8). Efrapeptin appears to inhibit F1-ATPase by blocking the conversion of subunit beta E to a nucleotide binding conformation, as would be required by an enzyme mechanism involving cyclic interconversion of catalytic sites.

Effects of receptor dimerization on the interaction between the class I major histocompatibility complex-related Fc receptor and IgG.

The neonatal Fc receptor (FcRn) transports maternal IgG from ingested milk in the gut to the bloodstream of newborn mammals. An FcRn dimer was observed in crystals of the receptor alone and of an FcRn-Fc complex, but its biological relevance was unknown. Here we use surface plasmon resonance-based biosensor assays to assess the role of FcRn dimerization in IgG binding. We find high-affinity IgG binding when FcRn is immobilized on a biosensor chip in an orientation facilitating dimerization but not when its orientation disrupts dimerization. This result supports a model in which IgG-induced dimerization of FcRn is relevant for signaling the cell to initiate endocytosis of the IgG-FcRn complex.

An A-DNA triplet code: thermodynamic rules for predicting A- and B-DNA.

The ability to predict macromolecular conformations from sequence and thermodynamic principles has long been coveted but generally has not been achieved. We show that differences in the hydration of DNA surfaces can be used to distinguish between sequences that form A- and B-DNA. From this, a "triplet code" of A-DNA propensities was derived as energetic rules for predicting A-DNA formation. This code correctly predicted > 90% of A- and B-DNA sequences in crystals and correlates with A-DNA formation in solution. Thus, with our previous studies on Z-DNA, we now have a single method to predict the relative stability of sequences in the three standard DNA duplex conformations.

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na+…O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na+ by K+, Rb+ or Cs+ and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb+ or Cs+ also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 Å.

Truncated hexa-octahedral magnetite crystals in ALH84001: Presumptive biosignatures

McKay et al. [(1996) Science 273, 924–930] suggested that carbonate globules in the meteorite ALH84001 contained the fossil remains of Martian microbes. We have characterized a subpopulation of magnetite (Fe3O4) crystals present in abundance within the Fe-rich rims of these carbonate globules. We find these Martian magnetites to be both chemically and physically identical to terrestrial, biogenically precipitated, intracellular magnetites produced by magnetotactic bacteria strain MV-1. Specifically, both magnetite populations are single-domain and chemically pure, and exhibit a unique crystal habit we describe as truncated hexa-octahedral. There are no known reports of inorganic processes to explain the observation of truncated hexa-octahedral magnetites in a terrestrial sample. In bacteria strain MV-1 their presence is therefore likely a product of Natural Selection. Unless there is an unknown and unexplained inorganic process on Mars that is conspicuously absent on the Earth and forms truncated hexa-octahedral magnetites, we suggest that these magnetite crystals in the Martian meteorite ALH84001 were likely produced by a biogenic process. As such, these crystals are interpreted as Martian magnetofossils and constitute evidence of the oldest life yet found.

Chains of magnetite crystals in the meteorite ALH84001: Evidence of biological origin

The presence of magnetite crystal chains, considered missing evidence for the biological origin of magnetite in ALH84001 [Thomas-Keprta, K. L., Bazylinski, D. A., Kirschvink, J. L., Clemett, S. J., McKay, D. S., Wentworth, S. J., Vali, H., Gibson, E. K., Jr., & Romanek, C. S. (2000) Geochim. Cosmochim. Acta 64, 4049–4081], is demonstrated by high-power stereo backscattered scanning electron microscopy. Five characteristics of such chains (uniform crystal size and shape within chains, gaps between crystals, orientation of elongated crystals along the chain axis, flexibility of chains, and a halo that is a possible remnant of a membrane around chains), observed or inferred to be present in magnetotactic bacteria but incompatible with a nonbiological origin, are shown to be present. Although it is unlikely that magnetotactic bacteria were ever alive in ALH84001, decomposed remains of such organisms could have been deposited in cracks in the rock while it was still on the surface on Mars.

Significance of bound water to local chain conformations in protein crystals.

We examine how the polypeptide chain in protein crystal structures exploits the multivalent hydrogen-bonding potential of bound water molecules. This shows that multiple interactions with a single water molecule tend to occur locally along the chain. A distinctive internal-coordinate representation of the local water-binding segments reveals several consensus conformations. The fractional water occupancy of each was found by comparison of the total number of conformations in the database regardless of the presence or absence of bound water. The water molecule appears particularly frequently in type II beta-turn geometries and an N-terminal helix feature. This work constitutes a first step into assessing not only the generality but also the significance of specific water binding in globular proteins.

The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.