Rik K. Wierenga

Prediction of the occurrence of the ADP-binding βαβ-fold in proteins, using an amino acid sequence fingerprint

Abstract An amino acid sequence “fingerprint” has been derived that can be used to test if a particular sequence will fold into aβαβ-unit with ADP-binding properties. It was deduced from a careful analysis of the known three-dimensional structures of ADP-binding βαβ-folds. This fingerprint is in fact a set of 11 rules describing the type of amino acid that should occur at a specific position in a peptide fragment. The total length of this fingerprint varies between 29 and 31 residues. By checking against all possible sequences in a database, it appeared that every peptide, which exactly follows this fingerprint, does indeed fold into an ADP-binding βαβ-unit.

Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 Å resolution: analysis of the enzyme-substrate and enzyme-product complexes

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)

Insights into Src kinase functions: structural comparisons

Recent structures of Src tyrosine kinases reveal complex mechanisms for regulation of enzymatic activity. The regulatory SH3 and SH2 domains bind to the back of the catalytic kinase domain via a linker region that joins the SH2 domain to the catalytic domain. Members of a subgroup of the Src kinase family show distinct features in this linker and in the loops that interact with it. Hydrophobicity of key residues in this region is important for intramolecular regulation. The kinases Abl, Btk and Csk seem to have the same molecular architecture as Src. Structural comparisons between serine/threonine and tyrosine kinases indicate a specific twisting mechanism involving the N- and C-terminal lobes of the catalytic domain. This motion could provide insights into the various mechanisms used to regulate kinase activity.

The 2.8å Crystal Structure of peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae : a five-layered αβαβα structure constructed from two core domains of identical topology

Abstract Background The peroxisomal enzyme 3-ketoacyl-coenzyme A thiolase of the yeast Saccharomyces cerevisiae is a homodimer with 417 residues per subunit. It is synthesized in the cytosol and subsequently imported into the peroxisome where it catalyzes the last step of the β -oxidation pathway. We have determined the structure of this thiolase in order to study the reaction mechanism, quaternary associations and intracellular targeting of thiolases generally, and to understand the structural basis of genetic disorders associated with human thiolases. Results Here we report the crystal structure of unliganded yeast thiolase refined at 2.8A resolution. The enzyme comprises three domains; two compact core domains having the same fold and a loop domain. Each of the two core domains is folded into a mixed five-stranded β - sheet covered on each side by helices and the two are assembled into a five-layered α β α β structure. The central layer is formed by two helices, which point with their amino termini towards the active site. The loop domain, which is to some extent stabilized by interactions with the other subunit, runs over the surface of the two core domains, encircling the active site of its own subunit. Conclusions The crystal structure of thiolase shows that the active site is a shallow pocket, shaped by highly conserved residues. Two conserved cysteines and a histidine at the floor of this pocket probably play key roles in the reaction mechanism. The two active sites are on the same face of the dimer, far from the amino and carboxyl termini of both subunits and the disordered amino-terminal import signal sequence.

Structure determination of the glycosomal triosephosphate isomerase from Trypanosoma brucei brucei at 2.4 A resolution.

The three-dimensional crystal structure of the enzyme triosephosphate isomerase from the unicellular tropical blood parasite Trypanosoma brucei brucei has been determined at 2.4 A resolution. This triosephosphate isomerase is sequestered in the glycosome, a unique trypanosomal microbody of vital importance for the energy-generating machinery of the trypanosome. The crystals contain one dimer per asymmetric unit. The structure could be solved by the method of molecular replacement, using the refined co-ordinates of chicken triosephosphate isomerase as a search model. The positions and individual isotropic temperature factors of the 3792 atoms of the complete dimer have been refined by the Hendrickson & Konnert restrained refinement procedure. While tight restraints have been maintained on the bonded distances, the R-factor has dropped to 23.2% for 12317 reflections between 6 A and 2.4 A. A total of 0.6 mg of enzyme was used for establishing the correct crystallization conditions and solving the three-dimensional structure. Although the sequences of trypanosomal and chicken triosephosphate isomerase are identical at only 52% of the 247 common positions, the overall folds are very similar. The architecture of the active sites is virtually the same with 85% of the side-chains being identical. On the other hand, the residues involved in the dimer contacts are the same at only 55% of the positions. Nevertheless, the position of the local 2-fold axis in the chicken and glycosomal dimers is similar. A remarkable feature of glycosomal triosephosphate isomerase is its high overall positive charge. This extra charge is concentrated in four clusters of positively charged side-chains on the surface of the dimer, quite far away from the active site. These clusters may be involved in the mechanism of import of this triosephosphate isomerase into the glycosome.

The crystal structure of human CskSH3: structural diversity near the RT-Src and n-Src loop

SH3 domains are modules occurring in diverse proteins, ranging from cytoskeletal proteins to signaling proteins, such as tyrosine kinases. The crystal structure of the SH3 domain of Csk (c‐Src specific tyrosine kinase) has been refined at a resolution of 2.5 Å, with an R‐factor of 22.4%. The structure is very similar to the FynSHS crystal structure. When comparing CskSHS and FynSH3 it is seen that the structural and charge differences of the RT‐Src loop and the n‐Src loop, near the conserved Trp47, correlate with different binding properties of these SH3 domains. The structure comparison suggests that those glycines and acid residues which are very well conserved in the SH3 sequences are important for the stability of the SH3 fold.

Crystallographic binding studies with triosephosphate isomerases: Conformational changes induced by substrate and substrate‐analogues

TIM catalyses the interconversion of a triosephosphate aldehyde into a triosephosphate ketone. This is a simple chemical reaction in which only protons are transferred, The crystallographic studies of TIM from chicken, yeast and trypanosome complexed with substrate and substrate analogues are discussed. The substrate binds in a deep pocket. On substrate binding, large conformational changes are induced in three loops. As a result of these conformational changes in the liganded Structure, the active site pocket is sealed off from bulk solvent and the sidechain of the catalytic glutamate becomes optimally positioned for catalysis.

Characterization of the genes for fructose-bisphosphate aldolase in Trypanosoma brucei

In Trypanosoma brucei stock 427 the glycolytic enzyme fructose-bisphosphate aldolase is encoded by two tandemly linked genes of identical sequence. Such a tandem arrangement of aldolase genes is also present in other T. brucei stocks of unrelated origin. In stock 427 one of the allelic genes is a pseudogene, as a result of a one-nucleotide deletion. The genes code for a polypeptide of 371 amino acids, with a calculated molecular weight of 40,940. The protein that is predicted from the gene sequence has 45-48% positional identity with known aldolase sequences of other organisms. The trypanosomal protein is, however, unique in having a 10 amino-acid insertion near its N-terminus and high number of basic residues, a feature it shares with other glycolytic enzymes of T. brucei. These glycolytic enzymes have in common that they are located in microbody-like organelles, the glycosomes. We have previously proposed that the positively charged residues may be involved in the import of the proteins into the organelles.

Three new crystal structures of point mutation variants of mono TIM: conformational flexibility of loop-1, loop-4 and loop-8

BACKGROUNDnWild-type triosephosphate isomerase (TIM) is a very stable dimeric enzyme. This dimer can be converted into a stable monomeric protein (monoTIM) by replacing the 15-residue interface loop (loop-3) by a shorter, 8-residue, loop. The crystal structure of monoTIM shows that two active-site loops (loop-1 and loop-4), which are at the dimer interface in wild-type TIM, have acquired rather different structural properties. Nevertheless, monoTIM has residual catalytic activity.nnnRESULTSnThree new structures of variants of monoTIM are presented, a double-point mutant crystallized in the presence and absence of bound inhibitor, and a single-point mutant in the presence of a different inhibitor. These new structures show large structural variability for the active-site loops, loop-1, loop-4 and loop-8. In the structures with inhibitor bound, the catalytic lysine (Lys13 in loop-1) and the catalytic histidine (His95 in loop-4) adopt conformations similar to those observed in wild-type TIM, but very different from the monoTIM structure.nnnCONCLUSIONSnThe residual catalytic activity of monoTIM can now be rationalized. In the presence of substrate analogues the active-site loops, loop-1, loop-4 and loop-8, as well as the catalytic residues, adopt conformations similar to those seen in the wild-type protein. These loops lack conformational flexibility in wild-type TIM. The data suggest that the rigidity of these loops in wild-type TIM, resulting from subunit-subunit contacts at the dimer interface, is important for optimal catalysis.

An interface point-mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations

Wild‐type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His‐47 of wtTIM has been mutated into an asparagine. Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near‐UV and far‐UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give T m‐values of 41°C for wtTIM, whereas the T m‐value for the monomeric form of H47N is approximately 7°C lower.