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Biomedical subjects

R M Garavito

Publications and source records attributed to R M Garavito.

At least 19 recordsLinked to original sources
PubMed1998

The structure of mammalian hexokinase-1.

We have determined the structures of the glucose-6-phosphate (G6P)-inhibitable 100,000 Mr Type I hexokinase from rat and the G6P-sensitive 50,000 Mr hexokinase from Schistosoma mansoni at a resolution of 2.8 and 2.6 A respectively. The structures define the glucose and G6P binding sites in these enzymes, suggest the mechanisms of intradomain G6P inhibition and activity loss in the Type I hexokinase N-terminal half, and reveal the structure of the membrane targeting motif that integrates the Type I hexokinase into the outer mitochondrial membrane.

Animals↗
PubMed1996

Synthesis and use of iodinated nonsteroidal antiinflammatory drug analogs as crystallographic probes of the prostaglandin H2 synthase cyclooxygenase active site.

The cyclooxygenase activity of the membrane protein prostaglandin H2 synthase isoform 1 (PGHS-1) is the target of the nonsteroidal antiinflammatory drugs (NSAIDs). The X-ray crystal structures of PGHS-1 in complex with the NSAIDs flurbiprofen and bromoaspirin have been determined previously [Picot, D., et al. (1994) Nature 367, 243-249; Loll, P. J., et al. (1995) Nat. Struct. Biol. 2, 637-643]. We report here the preparation and characterization of novel potent iodinated analogs of the NSAIDs indomethacin and suprofen, as well as the refined X-ray crystal structures of their complexes with PGHS-1. The PGHS-iodosuprofen complex structure has been refined at 3.5 A to an R-value of 0.189 and shows the suprofen analog to share a common mode of binding with flurbiprofen. The PGHS-iodoindomethacin complex structure has been refined at 4.5 A to an R-value of 0.254. The low resolution of the iodoindomethacin complex structure precludes detailed modeling of drug-enzyme interactions, but the electron-dense iodine atom of the inhibitor has been unambiguously located, allowing for the placement and approximate orientation of the inhibitor in the enzyme's active site. We have modeled two equally likely binding modes for iodoindomethacin, corresponding to the two principal conformers of the inhibitor. Like flurbiprofen, iodosuprofen and iodoindomethacin bind at the end of the long channel which leads into the enzyme active site. Binding at this site presumably blocks access of substrate to Tyr-385, a residue essential for catalysis. No evidence is seen for significant protein conformational differences between the iodoindomethacin and iodosuprofen of flurbiprofen complex structures.

Animals↗
PubMed1996

2-bromoacetoxybenzoic acid, a brominated aspirin analog.

The crystal structure of 2-bromoacetoxybenzoic acid, C9H7BrO4, shows it to be a close structural analog of aspirin. The carboxylic acid moiety is twisted by 7.7 (4) degrees out of the plane of the aromatic ring. The acetyl group, like that of aspirin, shows bond-angle distortions from ideal values while remaining essentially planar. The Br atom is rotationally disordered and has been modeled as occupying two sites related by a 13 (1) degree rotation about the C8--C9 bond.

Aspirin↗
PubMed1996

1-(4-iodobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid, an iodinated indomethacin analog.

The crystal structure of 1-(4-iodobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid, C19H16INO4, an analog of indomethacin, is reported. Bond distances and angles in the title compound closely resemble those reported for indomethacin and reflect the presence of steric strain at the site of the linkage between the 4-iodobenzoyl group and the indole moiety. The orientation of the 4-iodobenzoyl group with respect to the indole ring is not the same in the title compound as it is in indomethacin; the two structures are related by a rotation of 186 degrees about the C2--N1--C10--C11 torsion angle.

Crystallography, X-Ray↗
PubMed1996

A novel, multilayer structure of a helical peptide.

X-ray diffraction analysis at 1.5 A resolution has confirmed the helical conformation of a de novo designed 18-residue peptide. However, the crystal structure reveals the formation of continuous molecular layers of parallel-packed amphiphilic helices as a result of much more extensive helix-helix interactions than predicted. The crystal packing arrangement, by virtue of distinct antiparallel packing interactions, segregates the polar and apolar surfaces of the helices into discrete and well-defined interfacial regions. An extensive "ridges-into-grooves" interdigitation characterizes the hydrophobic interface, whereas an extensive network of salt bridges and hydrogen bonds dominates the corresponding hydrophilic interface.

Amino Acid Sequence↗
PubMed1996

Strategies for crystallizing membrane proteins.

Crystallizing membrane proteins remains a challenging endeavor despite the increasing number of membrane protein structures solved by X-ray crystallography. The critical factors in determining the success of the crystallization experiments are the purification and preparation of membrane protein samples. Moreover, there is the added complication that the crystallization conditions must be optimized for use in the presence of detergents although the methods used to crystallize most membrane proteins are, in essence, straightforward applications of standard methodologies for soluble protein crystallization. The roles that detergents play in stability and aggregation of membrane proteins as well as the colloidal properties of the protein-detergent complexes need to be appreciated and controlled before and during the crystallization trials. All X-ray quality crystals of membrane proteins were grown from preparations of detergent-solubilized protein, where the heterogeneous natural lipids from the membrane have been replaced by a homogeneous detergent environment. It is the preparation of such monodisperse, isotropic solutions of membrane proteins that has allowed the successful application of the standard crystallization methods routinely used on soluble proteins. In this review, the issues of protein purification and sample preparation are addressed as well as the new refinements in crystallization methodologies for membrane proteins. How the physical behavior of the detergent, in the form of micelles or protein-detergent aggregates, affects crystallization and the adaptation of published protocols to new membrane protein systems are also addressed. The general conclusion is that many integral membrane proteins could be crystallized if pure and monodisperse preparations in a suitable detergent system can be prepared.

Animals↗
PubMed1995

The structure of OmpF porin in a tetragonal crystal form.

BACKGROUND: OmpF porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules, such as nutrients and waste products, across the outer membrane of Escherichia coli. Very few membrane proteins have been crystallized in three dimensions, yet this stable protein can be obtained in several crystal forms. Comparison of the structures of the same membrane protein in two different packing environments is of major interest, because it allows us to explore the integrity of the structure outside the natural membrane environment. RESULTS: The structure of OmpF porin in a tetragonal crystal form with two trimers per asymmetric unit has been determined at 3.2 A resolution and compared with that obtained previously in a trigonal crystal form. The lattice contacts involve only polar atoms, whereas extensive hydrophobic protein-protein interactions were found in the trigonal lattice. The trimer structure is virtually identical in both. CONCLUSIONS: Our comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints and, thus, presumably bears close resemblance to the in vivo structure. The tetragonal crystal structure has provided the starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article.

Amino Acid Sequence↗
PubMed1995

Detergent structure in tetragonal crystals of OmpF porin.

BACKGROUND: The high-resolution structures of five porins have been solved by X-ray crystallography including the trigonal crystal form of the trimeric OmpF porin from Escherichia coli. In an accompanying article, the structure of the tetragonal form of OmpF porin is presented. In contrast to the trigonal crystal form, the protein surfaces normally in contact with lipids in the membrane are exposed and interact with amphiphiles in the tetragonal crystal. Thus, the tetragonal form can be used to investigate protein-detergent interactions. RESULTS: Using single-crystal neutron diffraction studies and two different detergents (one of them deuterated in its hydrophobic moiety), details of the amphiphile-protein interactions are revealed. Detergent molecules bind to the so-called hydrophobic zone that surrounds the OmpF porin trimer and which is exposed to lipid in the native environment. The aromatic rings on both sides of the hydrophobic zone coincide with the boundary between non-polar and polar moieties of the detergents. CONCLUSIONS: In the tetragonal crystal form of OmpF porin, the membrane-exposed area is accessible from the aqueous solution. It is coated by a film of detergent molecules, which presumably mimics the interactions of the protein with lipids in the biological membrane. In the trigonal form, protein-protein interactions predominate in the hydrophobic zone. These may reflect the tight interactions between trimers that are observed in the biological membrane.

Bacterial Outer Membrane Proteins↗
PubMed1995

X-ray crystal structures of staphylococcal nuclease complexed with the competitive inhibitor cobalt(II) and nucleotide.

Two crystal structures of ternary complexes of staphylococcal nuclease, cobalt(II), and the mononucleotide pdTp are reported. The first has been refined at 1.7 A to a crystallographic R value of 0.198; the second, determined from a crystal soaked for 9 months in a slightly different mother liquor than the first crystal, has been refined at 1.85 A to an R value of 0.174. In the first structure, the cobalt ion is displaced 1.94 A from the normal calcium position, and the active site is dominated by a salt bridge between Asp-21 and Lys-70 from a symmetry-related molecule in the crystal lattice. The Co2+ ion appears unable to displace this lysine; consequently, the metal is bound in a vestibular site adjacent to the calcium site. The metal-binding pocket in the second structure adopts a configuration similar to that of the calcium complex, with the cobalt ion binding only 0.36 A from the calcium position. However, an inner sphere water seen in the calcium structure is missing from this structure. The cobalt ion in the second structure appears to be loosely or transiently coordinated within the calcium binding pocket, as evidenced by the high value of its refined thermal factor. Loss of catalytic activity for cobalt(II)-substituted nuclease is perhaps due to its inability to bind this inner sphere water.

Binding Sites↗
PubMed1995

An examination of the source of the tyrosyl radical in ovine prostaglandin endoperoxide synthase-1.

A tyrosyl radical, which may initiate the cyclooxygenase reaction, has been detected in prostaglandin H synthase by electron paramagnetic resonance spectroscopy. In the crystal structure of ovine prostaglandin H synthase-1, Tyr348 and Tyr385 are in close proximity to the heme. We mutated these residues to phenylalanine to test for their involvement in tyrosyl radical formation. Native enzyme formed a tyrosyl radical centered at g = 2.0036 with a width of 28 gauss. The Y348F mutant formed a singlet signal similar to that of native enzyme with a width of 28 gauss (g = 2.0039). In contrast, the radical signals seen with the Y385F and Y348F/Y385F mutants were 23 gauss (g = 2.004) and 22 gauss (g = 2.0037). In short, tyrosyl radicals are formed even in the absence of both Tyr348 and Tyr385. In Y345F containing mutants, a cluster of aromatic amino acids which surrounds the heme group may provide an alternate pathway for electron abstraction from a more distant tyrosine, yielding a narrow tyrosyl radical signal.

Amino Acid Sequence↗
PubMed1995

Calibration of size-exclusion chromatography: use of a double Gaussian distribution function to describe pore sizes.

A method for the calibration of size-exclusion chromatographic columns is proposed that takes into account the nonlinear dependence of the Stokes radius Rs upon the partition coefficient KD. The method is based on the assumption that the pore size distribution of a molecular sieve column can be described by the sum of two Gaussian distributions and has been successfully tested with low-pressure chromatography gels (Sephacryl and Superose) and HPLC gels (TSK SW) over a wide range of protein sizes. An application of this method is described, in which aggregation states of the membrane protein prostaglandin H2 synthase solubilized in nonionic detergents are estimated.

Calibration↗
PubMed1995

The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2 synthase.

Aspirin exerts its anti-inflammatory effects through selective acetylation of serine 530 on prostaglandin H2 synthase (PGHS). Here we present the 3.4 A resolution X-ray crystal structure of PGHS isoform-1 inactivated by the potent aspirin analogue 2-bromoacetoxy-benzoic acid. Acetylation by this analogue abolishes cyclooxygenase activity by steric blockage of the active-site channel and not through a large conformational change. We observe two rotameric states of the acetyl-serine side chain which block the channel to different extents, a result which may explain the dissimilar effects of aspirin on the two PGHS isoforms. We also observe the product salicylic acid binding at a site consistent with its antagonistic effect on aspirin activity.

Acetylation↗
PubMed1994

Prostaglandin H synthase: implications for membrane structure.

The crystal structure of the membrane protein prostaglandin H synthase (PGHS) provides strong evidence for the existence of monotopic membrane proteins: PGHS seems to interact with the membrane via a motif of amphipathic helices positioned parallel to the plane of the membrane. The orientation of this unique membrane binding motif is fixed in space by an epidermal growth factor(EGF)-like module on its amino-terminal end and by the catalytic domain at its carboxy-terminal end. The catalytic domain of PGHS has a high structural homology to other mammalian heme peroxidases.

Amino Acid Sequence↗