Biomedical subjects
Publications and source records attributed to Russell F Doolittle.
Although it has long been realized that a large portion of the fibrinogen alpha chain has little if any defined structure, the physiological significance of this flexible appendage remains mysterious.
Synthetic peptides corresponding to the amino-terminal sequence of the beta chain of fibrin increase the turbidity of fibrin clots, whether they are generated by the direct interaction of thrombin and fibrinogen or by the reassociation of fibrin monomers. The turbidity of batroxobin-induced clots, which are characteristically "fine," is increased even more dramatically. Pentapeptides are more effective than tetrapeptides. Surprisingly, the same peptides also delay fibrinolysis, whether activated by exogenously added plasmin or from the fibrin-enhanced stimulation of tissue plasminogen activator (tPA) activation of plasminogen. The peptides have only a very slight effect on the plasmic hydrolysis of a chromogenic peptide, either by the direct addition of plasmin or by plasmin generated from plasminogen by tPA. The synthetic peptides mimicking the B knobs appear to exert their action in two ways. First, they render fibrin less vulnerable to attack by plasmin. Second, they delay the fibrin activation of tPA. The latter is attributed to their ability to prevent the binding of the authentic B knob, which itself is located at the end of a flexible 50-residue tether and which needs time to find its elusive "hole". We propose that, when after a while the tethered knob does become inserted, it locks the betaC domain in a conformation that allows access to tPA-plasminogen-binding sites, whereas the untethered synthetic knobs restrict the fibrin to a conformation in which those sites remain inaccessible. Thus, although the interaction involving the A knob and gammaC hole is the basis for the polymerization of fibrin, the comparable but delayed interaction involving the B knob and the betaC hole is ultimately directed at preparing the clot for its eventual destruction.
The 420 kDa hemolymph clottable protein has been purified from the California spiny lobster Panulirus interrruptus and crystallized in both monoclinic and orthorhombic space groups. Complete data sets have been collected from the two crystal forms to a maximum resolution of 3.9 and 3.2 A, respectively. The monoclinic crystals exhibit unusual noncrystallographic symmetry with pseudo-ninefold rotational symmetry generated from three dimers per asymmetric unit. The orthorhombic crystals have a single dimer per asymmetric unit. Attempts to phase the data are currently under way.
3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS), EC 4.1.3.5, is an essential enzyme in rubber biosynthesis in Hevea brasiliensis. We have isolated a new cDNA encoding HMGS in H. brasiliensis. The full-length hmgs2 consists of 1,916-bp and encodes a protein of 464 amino acids with a predicted molecular mass of 51.27 kDa and an isoelectric point of 6.02. In comparison, HMGS1 and HMGS2 show 92% and 94% nucleotide and amino acid sequence identities, respectively. Semiquantitative RT-PCR analysis indicates that the hmgs2 is more highly expressed in laticifer and petiole than in leaves. Sequence searching and alignment revealed that HMGS is a distant relative of the condensing enzyme; beta-ketoacyl acyl carrier protein synthase III (ACP synthase III), EC 2.3.1.41, identified three completely conserved residues; Cys(117), His(247), and Asn(326). The relationship was greatly strengthened by making a proper alignment of numerous sequences of both HMGS and ACP synthase III. The same Cys(117), His(247), and Asn(326) absolutely conserved in both groups play a catalytic role in ACP synthase III, while such a role of Cys and His has only been reported for HMGS. According to site-directed mutagenesis, the expressed wild-type enzyme shows comparable level with mutant proteins. The mutation of Cys(117) and Asn(326) affects the HMGS activity, indicating that Cys(117) and Asn(326) are important amino acids for the catalytic activity of HMGS. A phylogenetic tree constructed on the basis of proper multiple alignment indicates that HMGS1 and HMGS2 result from recent gene duplication. This is also the case for HMGS and ACP synthase III, which appear to have arisen from an ancient gene duplication event of an ancestral condensing enzyme. Therefore, a possible secondary structure of HMGS could be predicted based on the Protein Data Bank information of ACP synthase III.
A simple classification scheme that uses only the presence or absence of a protein domain architecture has been used to determine the phylogeny of 174 complete genomes. The method correctly divides the 174 taxa into Archaea, Bacteria, and Eukarya and satisfactorily sorts most of the major groups within these superkingdoms. The most challenging problem involved 119 Bacteria, many of which have reduced genomes. When a weighting factor was used that takes account of difference in genome size (number of considered folds), small-genome taxa were mostly grouped with their full-sized counterparts. Although not every organism appears exactly at its classical phylogenetic position in these trees, the agreement appears comparable with the efforts of others by using sophisticated sequence analysis and/or combinations of gene content and gene order. During the course of the study, it emerged that there is a core set of approximately 50 folds that is found in all 174 genomes and a single fold diagnostic of all Archaea.
A decade of access to whole-genome sequences has been increasingly revealing about the informational network relating all living organisms. Although at one point there was concern that extensive horizontal gene transfer might hopelessly muddle phylogenies, it has not proved a severe hindrance. The melding of sequence and structural information is being used to great advantage, and the prospect exists that some of the earliest aspects of life on Earth can be reconstructed, including the invention of biosynthetic and metabolic pathways. Still, some fundamental phylogenetic problems remain, including determining the root--if there is one--of the historical relationship between Archaea, Bacteria and Eukarya.
In this brief memoir, I reflect on the great insight John Ferry exhibited in his extremely influential 1952 paper on the mechanism of fibrinogen being changed into fibrin.
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The blood coagulation scheme for the puffer fish, Fugu rubripes, has been reconstructed on the basis of orthologs of genes for mammalian blood clotting factors being present in its genome. As expected, clotting follows the same fundamental pattern as has been observed in other vertebrates, even though genes for some clotting factors found in mammals are absent and some others are present in more than one gene copy. All told, 26 different proteins involved in clotting or fibrinolysis were searched against the puffer fish genome. Of these, orthologs were found for 21. Genes for the "contact system" factors (factor XI, factor XII, and prekallikrein) could not be identified. On the other hand, two genes were found for factor IX and four for factor VII. It was evident that not all four factor VII genes are functional, essential active-site residues having been replaced in two of them. A search of the genome of a urochordate, the sea squirt, Ciona intestinalis, did not turn up any genuine orthologs for these 26 factors, although paralogs and/or constituent domains were evident for virtually all of them.
During the past several years, a number of crystal structures have been determined of fragments from fibrinogen and fibrin and, most recently, a structure of a native fibrinogen. One feature of the fibrinogen molecule that has emerged from these studies has to do with its "loose ends," segments of the molecule that are extremely mobile and not discernable by X-ray crystallography. Some, if not all, of this flexibility is functionally important. Small synthetic peptides based on mobile parts of fibrinogen exposed by the action of thrombin have contributed significantly to these studies and may yet prove useful therapeutically. In the end, although crystal structures have added greatly to our understanding of fibrin formation, much still needs to be unraveled about how clots form.
We have recently determined the structure of a native fibrinogen at 2.7-A resolution. Not the least of the hurdles during the many years of this project was growing X-ray-grade crystals from suitably purified proteins. Small, synthetic peptides based on the parts of fibrinogen exposed by the action of thrombin contributed greatly to these experiments. In addition, trimethylamine oxide (TMAO) was found to improve the diffraction of fibrinogen crystals. The history of my interest in fibrinogen and its crystallization can be traced back in part to some early interactions with John Edsall.
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The crystal structure of fragment double-D from factor XIII-cross-linked lamprey fibrin has been determined at 2.9 A resolution. The 180 kDa covalent dimer was cocrystallized with the peptide Gly-His-Arg-Pro-amide, which in many fibrinogens, but not that of lamprey, corresponds to the B-knob exposed by thrombin. The structure was determined by molecular replacement, a recently determined structure of lamprey fragment D being used as a search model. GHRPam was found in both the gamma- and beta-chain holes. Unlike the situation with fragment D, the crystal packing of the cross-linked double-D structure exhibits two different D-D interfaces, each gamma-chain facing gamma-chains on two other molecules. One of these (interface I) involves the asymmetric interface observed in all other D fragments and related structures. The other (interface II) encompasses a completely different set of residues. The two abutments differ in that interface I results in an "in line" arrangement of abutting molecules and the interface II in a "zigzag" arrangement. So far as can be determined (the electron density could only be traced on one side of the cross-links), it is the gamma-chains of the newly observed zigzag units (interface II) that are joined by the reciprocal epsilon-amino-gamma-glutamyl cross-links. Auspiciously, the same novel D-D interface was observed in two lower-resolution crystal structures of human double-D preparations that had been crystallized under unusual circumstances. These observations show that double-D structures are linked in a way that is sufficiently flexible to accommodate different D-D interfaces under different circumstances.
The crystal structure of fragment D from lamprey fibrinogen has been determined at 2.8 A resolution. The 89 kDa protein was cocrystallized with the peptide Gly-His-Arg-Pro-amide, which in many fibrinogens-but not lamprey-corresponds to the B knob exposed by thrombin. Because lamprey fragment D is more than 50% identical in sequence with human fragment D, the structure of which has been reported previously, it was possible to use the method of molecular replacement. The space group of the lamprey crystals is P1; there are four molecules in the unit cell. Although the fragments are packed head to head by the same D:D interface as is observed in other related preparations containing fragments D, the tails are uniquely joined by an unnatural association of the terminal sections of the residual coiled coils from adjacent molecules. Some features of the lamprey structure are clearer than have been observed in previous fragment D structures, including the beta-chain carbohydrate cluster, for one, and the important gamma-chain carboxyl-terminal segment, for another. The most significant differences between the lamprey and human structures occur in connecting loops at the entryways to the beta-chain and gamma-chain binding pockets.
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According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers1, 2 present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveal that eukaryotic actin and the bacterialhomolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.