Torben V. Borchert

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.

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

BACKGROUND Wild-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. RESULTS Three 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. CONCLUSIONS The 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.