Biomedical subjects

Mark Bycroft

Publications and source records attributed to Mark Bycroft.

25 records · Page 2Linked to original sources

PubMed2003

Crystal structure of the malignant brain tumor (MBT) repeats in Sex Comb on Midleg-like 2 (SCML2).

Sex Comb on Midleg (SCM) belongs to the Polycomb group of proteins, which are involved in transcriptional regulation in Drosophila. It is one of the components of Polycomb repressive complex 1, a multiprotein complex of Polycomb group proteins involved in the maintenance of repression and the blocking of chromatin remodeling. SCM contains two approximately 100-residue malignant brain tumor (MBT) repeats at the N terminus. These repeats are also found in other proteins involved in transcriptional repression. Here, we report the 1.78-A crystal structure of the two MBT repeats of SCM-like 2 (SCML2), a human homologue of SCM. Each repeat consists of an extended arm and a beta-barrel core. There are significant structural similarities to the Tudor, PWWP, and chromo domains, suggesting probable evolutionary relationships and functional similarities between the MBT repeats and these domains.

Amino Acid Sequence↗

PubMed2003

Structural variation in PWWP domains.

The PWWP domain is a ubiquitous eukaryotic protein module characterised by a region of sequence similarity of approximately 80 amino acids containing a highly conserved PWWP motif. It is frequently found in proteins associated with chromatin. We have determined the structure of a PWWP domain from the S. pombe protein SPBC215.07c using NMR spectroscopy. The structure is composed of a five stranded beta barrel followed by two alpha helices. Comparison to the recently reported structure of a homologous domain from the mammalian DNA methyltransferase Dnmt3b reveals substantial differences both in the C-terminal helical region and in the PWWP motif.

Amino Acid Sequence↗

PubMed2003

The crystal structure of AF1521 a protein from Archaeoglobus fulgidus with homology to the non-histone domain of macroH2A.

MacroH2A is an unusual histone H2A variant that has an extensive C-terminal tail that comprises approximately two thirds of the protein. The C-terminal non-histone domain of macroH2A is also found in a number of other proteins and has been termed the macro domain. Here we report the crystal structure to 1.7A of AF1521, a protein consisting of a stand-alone macro domain from Archaeoglobus fulgidus. The structure has a mixed alpha/beta fold that closely resembles the N-terminal DNA binding domain of the Escherichia coli leucine aminopeptidase PepA. The structure also shows some similarity to members of the P-loop family of nucleotide hydrolases.

Amino Acid Sequence↗

PubMed2003

Crystal structure of ribosomal protein L30e from the extreme thermophile Thermococcus celer: thermal stability and RNA binding.

We report here the high-resolution crystal structure of the ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer determined at cryo-temperature. When it is compared with its mesophilic homologue, L30e from yeast, a number of structural features that can enhance thermostability are revealed. Disordered residues corresponding to a large RNA-binding loop in yeast L30e are well structured in the T. celer protein. The overall charge of T. celer L30e is near neutral, whereas that of the yeast homologue is highly positive. This is the result of an increase in the number of acidic residues at the expense of polar residues, Asn, Ser, and Thr. Extensive ion pair networks are found on the molecular surface. Exposed nonpolar surface areas are reduced in the T. celer protein. Its side chain atoms preferably form hydrogen bonds with main chain atoms. Taken together, these factors contribute to high protein stability. The roles of well-conserved L30e residues are studied and found to be important in defining a very compact overall structure and in maintaining the structure of the RNA binding site. By comparing it with the yeast homologue, we also identified the residues that are responsible for RNA binding and built a model to illustrate how L30e binds to an RNA kink turn motif.

Amino Acids↗

PubMed2003

Solution structure and thermal stability of ribosomal protein L30e from hyperthermophilic archaeon Thermococcus celer.

To understand the structural basis of thermostability, we have determined the solution structure of a thermophilic ribosomal protein L30e from Thermococcus celer by NMR spectroscopy. The conformational stability of T. celer L30e was measured by guanidine and thermal-induced denaturation, and compared with that obtained for yeast L30e, a mesophilic homolog. The melting temperature of T. celer L30e was 94 degrees C, whereas the yeast protein denatured irreversibly at temperatures >45 degrees C. The two homologous proteins also differ greatly in their stability at 25 degrees C: the free energy of unfolding was 45 kJ/mole for T. celer L30e and 14 kJ/mole for the yeast homolog. The solution structure of T. celer L30e was compared with that of the yeast homolog. Although the two homologous proteins do not differ significantly in their number of hydrogen bonds and the amount of solvent accessible surface area buried with folding, the thermophilic T. celer L30e was found to have more long-range ion pairs, more proline residues in loops, and better helix capping residues in helix-1 and helix-4. A K9A variant of T. celer L30e was created by site-directed mutagenesis to examine the role of electrostatic interactions on protein stability. Although the melting temperatures of the K9A variant is approximately 8 degrees C lower than that of the wild-type L30e, their difference in T(m) is narrowed to approximately 4.2 degrees C at 0.5 M NaCl. This salt-dependency of melting temperatures strongly suggests that electrostatic interactions contribute to the thermostability of T. celer L30e.

Amino Acid Sequence↗

PubMed2002

Molecular mechanism of the interaction between MDM2 and p53.

We have investigated the kinetic and thermodynamic basis of the p53-MDM2 interaction using a set of peptides based on residues 15-29 of p53. Wild-type p53 peptide bound MDM2 with a dissociation constant of 580nM. Phosphorylation of S15 and S20 did not affect binding, but T18 phosphorylation weakened binding tenfold, indicating that phosphorylation of only T18 is responsible for abrogating p53-MDM2 binding. Truncation to residues 17-26 increased affinity 13-fold, but further truncation to 19-26 abolished binding. NMR studies of the binding of the p53-derived peptides revealed global conformational changes of the overall structure of MDM2, stretching far beyond the binding cleft, indicating significant changes in the domain dynamics of MDM2 upon ligand binding.

Calorimetry↗

PubMed2002

The structure of an FF domain from human HYPA/FBP11.

The FF domain is a 60 amino acid residue phosphopeptide-binding module found in a variety of eukaryotic proteins including the transcription elongation factor CA150, the splicing factor Prp40 and p190RHOGAP. We have determined the structure of an FF domain from HYPA/FBP11. The domain is composed of three alpha helices arranged in an orthogonal bundle with a 3(10) helix in the loop between the second and third alpha helices. The structure differs from those of other phosphopeptide-binding domains and represents a novel phosphopeptide-binding fold.

Amino Acid Sequence↗