Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase.

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.

Identification of the structural and functional domains of MutY, an Escherichia coli DNA mismatch repair enzyme.

The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26- and 13-kDa fragments. NH2-terminal sequencing showed that the p13 domain begins at Gln226, indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A.G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2'-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met1-Trp216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.

A conserved binding site on the receptor for polymeric Ig is homologous to CDR1 of Ig V kappa domains.

We have previously shown that the binding of human secretory component (SC) to polymeric IgA (PIgA) is inhibited by a synthetic peptide fragment of SC (SC[15-37]), and also by a mAb (mAb 6G11) which specifically recognizes the unbound form of SC and SC[15-37]. In this report we show that the binding of bovine, rabbit, and rat SC to human PIgA can also be inhibited by SC[15-37], and that the relative affinity of these three species of SC for human PIgA correlated with their relative affinity for mAb 6G11. The binding of mAb 6G11 to SC isolated from various species was especially affected by amino acid substitutions at position 26 of SC. Structural modeling revealed that the side chain of residue 26 is located on the external face of a loop in SC domain I which is homologous in size and position to the first complementarity determining region (CDR1) of Ig V kappa domains. However, the surface topography of the CDR1-like loop of SC is distinct from other known V kappa CDR1 loops due to the unique distribution of charged and bulky polar side chains which locate to the exterior face of the loop. Together, these findings indicated that the binding site on the receptor for PIg is highly conserved among species, and is composed, in part, of residues of domain I of SC which may form a distinct CDR1-like loop on the surface of the molecule.

Structure of antischistosome compounds. V 1,6-Hexanediylbis(triphenylphosphonium) dibromide.

C42H42P2(2+).2Br-, Mr = 768.6, triclinic, P1, a = 13.328 (2), b = 23.110 (3), c = 9.417 (1) A, alpha = 90.75 (1), beta = 109.62 (1), gamma = 91.23 (1) degrees, V = 2730.86 A3, Z = 3, Dx = 1.402 g cm3, graphite-monochromatized Cu K alpha radiation, lambda = 1.5418 A, mu = 52.0 cm-1, F(000) = 1182, T = 292 K. Final R = 0.051 for 5225 reflections with I greater than 3 sigma (I). Structure solved by Patterson methods and delta F syntheses. One molecule is located at the center of symmetry of the unit cell. The hexane moieties of all three molecules are in the trans extended conformation. However, one of the P-C-C-C torsion angles in the complete molecule in the asymmetric unit is 118.4 (6) degrees, the corresponding P-C-C-C torsion angle about the other P atom being 178.7 (5) degrees. This creates a conformational asymmetry in an otherwise symmetric molecule. This conformation permits the formation of a nearly square, planar, noncovalent interaction involving this P atom with one of the Br- ions and a symmetry-related Br--P interaction. The remaining P atoms and Br- ions are noncovalently linked to this square, approximately along the alpha axis. This noncovalent network is nearly parallel to the [011] plane, but does not extend beyond the [020] and [020] planes. The half-molecule in the asymmetric unit is approximately 45 degrees to the whole molecule, with two of its phenyl rings directed toward and perpendicular to one of the phenyl rings of the asymmetric triphenylphosphonium group of the whole molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of antischistosome compounds. IV. (3-Bromopropyl)triphenylphosphonium bromide.

C21H21BrP+.Br-, Mr = 464.2, monoclinic, P2(1)/c, a = 11.165(2), b = 10.160(2), c = 17.614(3)A, beta = 104.99(1) degree, V = 1930.08 A3, Z = 4, Dx = 1.597 g cm-3, graphite-monochromatized Cu K alpha radiation, lambda = 1.5418 A, mu = 61.6 cm-1, F(000) = 928, T = 292 K. Final R = 0.052 for 3255 observed reflections with I greater than 3 omega (I). Structure solved by direct methods. The bromopropyl moiety is in an extended conformation with the gauche+ C-C-C-Br torsion angle orienting the C-Br vector in a direction similar to the direction of the N-Br vector observed in the (2-aminoethyl)triphenylphosphonium bromide hydrobromide structure and the cyano vector in the (3-cyano-propyl)triphenylphosphonium bromide structure.

Structure of antischistosome compounds. III. (3-Cyanopropyl)triphenylphosphonium bromide.

C22H21NP+.Br-, Mr = 410.3, monoclinic, P2(1)/c, a = 11.284 (1), b = 10.236 (1), c = 17.392 (2) A, beta = 105.33 (45) degrees, V = 1937.37 A3, Z = 4, D chi = 1.407 g cm-3, graphite-monochromatized Cu K alpha radiation, lambda = 1.5418 A, mu = 37.1 cm-1, F(000) = 840, T = 292 K. Final R = 0.044 for 3335 observed reflections. Structure solved by direct methods. The cyanopropyl moiety is in an extended conformation. However, the C-C-C-C torsion angle in this group is gauche+, which points the cyano group in a direction similar to the direction of the N-Br1 vector observed in the (2-aminoethyl)triphenylphosphonium bromide hydrobromide structure and the C-Br vector in the (3-bromopropyl)triphenylphosphonium bromide structure. This suggests a basis for the biological activities of these three compounds.

Crystal and molecular structure of tert.-butyloxycarbonyl-L-prolyl-alpha-aminoisobutyryl-L-alanyl-alpha- aminoisobutyrate methyl ester.

Boc-Pro-Aib-Ala-Aib-OMe crystallizes in the orthorhombic space group P2(1)2(1)2 with cell dimensions a = 17.701 (3) A, b = 17.476 (4) A, c = 9.686 (2) A, V = 2996.3 A3. The first three residues form a single turn of a 3(10)-helix stabilized by two intramolecular hydrogen bonds. Comparison of the conformation of the methyl ester of the tetrapeptide with that of its benzyl ester shows differences in the individual torsion angles of up to 29 degrees, although the overall conformation is conserved.

Calcium phosphate crystal formation in Escherichia coli from human urine: an in vitro study.

Escherichia coli with no demonstrable urease activity was inoculated into filter sterilized urine obtained from a healthy volunteer subject with no history of stone disease and then incubated at 37 degrees C. Bacteria were recovered at intervals between 1 and 10 days. Urinary pH was stable as compared to control urines and spontaneous crystal precipitation was not noted in controls. Recovered organisms were analyzed by x-ray powder diffractometry. An uncharacterized mineral phase (UMP) was first evident after 6 days. Calcium phosphate in the form of brushite and hydroxyapatite was apparent at 7 and 10 days respectively. This suggests a role for bacteria in calcium phosphate crystal formation in urine apart from urease activity and may contribute to the calcium phosphate component of urinary calculi.

Crystallization and preliminary X-ray diffraction studies of methemoglobin Bart's.

Methemoglobin Bart's (gamma 4) purified from the blood of an alpha thalassemic neonate has been crystallized in a crystal form unique for hemoglobins. The space group is trigonal, P3121, or the enantiomorph P3221, a = b = 55.85 A, c = 159.10 A. There are three tetrameric molecules in the unit cell with two gamma-chains per asymmetric unit.

The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution.

The structure of cytochrome b562 from Escherichia coli has been determined at 2.5 A resolution by x-ray diffraction methods. Protein phases were computed by the single isomorphous replacement method with anomalous scattering measurements from the native and uranyl acetate-substituted crystals. The electron density was averaged about the noncrystallographic 2-fold axis relating 2 molecules in the triclinic unit cell. The protein consists of four nearly parallel alpha helices and represents a new class of cytochrome structure. The heme group is inserted between the helices near one end of the molecule with one heme face partially exposed to solvent. The two heme ligands are histidine and methionine. The 2 phenylalanines are packed internally near the heme group, and the 2 tyrosines are on the surface, also near the heme group. The folding of the protein resembles that of hemerythrin and tobacco mosaic virus protein and shows a different topology from that of cytochrome b5, cytochrome c, or the globins.