Biomedical subjects

Dmitry B Veprintsev

Publications and source records attributed to Dmitry B Veprintsev.

22 records · Page 2Linked to original sources

PubMed2003

Structural basis for Arl1-dependent targeting of homodimeric GRIP domains to the Golgi apparatus.

Golgins are large coiled-coil proteins that play a role in Golgi structure and vesicle traffic. The Arf-like GTPase Arl1 regulates the translocation of GRIP domain-containing golgins to Golgi membranes. We report here the 1.7 A resolution structure of human Arl1-GTP in a complex with the GRIP domain of golgin-245. The structure reveals that the GRIP domain consists of an S-shaped arrangement of three helices. The domain forms a homodimer that binds two Arl1-GTPs using two helices from each monomer. The structure is consistent with golgin-245 forming parallel coiled-coils and suggests how Arl1-GTP/GRIP complexes interact with Golgi membranes via the N termini of Arl1-GTP and the C-terminal tails of the GRIP domains. In cells, bivalent association with Arl1-GTP would increase residence time of the golgins on Golgi membranes. Despite no conservation of sequence, topology, or even helical direction, several other effectors form similar interactions with small GTPases via a pair of alpha helices, suggesting a common structural basis for effector recognition.

ADP-Ribosylation Factors↗

PubMed2002

CRINEPT-TROSY NMR reveals p53 core domain bound in an unfolded form to the chaperone Hsp90.

The molecular chaperone Hsp90 sequesters oncogenic mutants of the tumor suppressor p53 that have unstable core domains. It is not known whether p53 is bound in an unfolded, partly folded, or distorted structure, as is unknown for the structure of any bound substrate of Hsp90. It is a particularly difficult problem to analyze in detail the structures of large complexes in which one component is (partly) unfolded. We have shown by transverse relaxation-optimized NMR spectroscopy combined with cross-correlated relaxation-enhanced polarization transfer (CRINEPT-TROSY) that p53 core domain bound in an approximately 200-kDa complex with Hsp90 was predominantly unfolded lacking helical or sheet secondary structure. This mode of binding might be a general feature of substrates of Hsp90.

Binding Sites↗

PubMed2002

Recognition of DNA by p53 core domain and location of intermolecular contacts of cooperative binding.

We present an analysis by NMR of a 58 kDa complex of the core domain of the tumour suppressor p53 with DNA that complements and extends the crystal structure analysis. Binding of specific DNA caused significant chemical shifts of residues on the DNA-binding interface that translated into the beta-sheet of the protein. Binding of non-specific DNA caused weak but qualitatively the same shifts, corresponding to weaker binding interactions. The observed chemical shift differences correlate with frequency of cancer-inducing mutations, suggesting that the affected residues contribute to the stability of p53 core domain-DNA complex. We also identified two affected regions on the surface of the protein: helix 1 (residues V173-C182) plus G244 and residues L114-T118, which may represent a dimerisation interface.

Allosteric Site↗

PubMed2002

A peptide that binds and stabilizes p53 core domain: chaperone strategy for rescue of oncogenic mutants.

Conformationally compromised oncogenic mutants of the tumor suppressor protein p53 can, in principle, be rescued by small molecules that bind the native, but not the denatured state. We describe a strategy for the rational search for such molecules. A nine-residue peptide, CDB3, which was derived from a p53 binding protein, binds to p53 core domain and stabilizes it in vitro. NMR studies showed that CDB3 bound to p53 at the edge of the DNA binding site, partly overlapping it. The fluorescein-labeled peptide, FL-CDB3, binds wild-type p53 core domain with a dissociation constant of 0.5 microM, and raises the apparent melting temperatures of wild-type and a representative oncogenic mutant, R249S core domain. gadd45 DNA competes with CDB3 and displaces it from its binding site. But this competition does not preclude CDB3 from being a lead compound. CDB3 may act as a "chaperone" that maintains existing or newly synthesized destabilized p53 mutants in a native conformation and then allows transfer to specific DNA, which binds more tightly. Indeed, CDB3 restored specific DNA binding activity to a highly destabilized mutant I195T to close to that of wild-type level.

Amino Acid Sequence↗