Kevin J. Lumb

Random-coil chemical shifts of phosphorylated amino acids

The 1H, 13C, 15N and 31 P random-coil chemical shifts and phosphate pKa values of the phosphorylated amino acids pSer, pThr and pTyr in the protected peptide Ac-Gly-Gly-X-Gly-Gly-NH2 have been obtained in water at 25 °C over the pH range 2 to 9. Analysis of ROESY spectra indicates that the peptides are unstructured. Phosphorylation induces changes in random-coil chemical shifts, some of which are comparable to those caused by secondary structure formation, and are therefore significant in structural analyses based on the chemical shift.

Intrinsic structural disorder and sequence features of the cell cycle inhibitor p57Kip2

The cell cycle inhibitor p57Kip2 induces cell cycle arrest by inhibiting the activity of cyclin‐dependent kinases. p57, although active as a cyclin A‐CDK2 inhibitor, is largely unfolded or intrinsically disordered as shown by circular dichroism and fluorescence spectra characteristic of an unfolded protein and a hydrodynamic radius consistent with an unfolded structure. In addition, the N‐terminal domain of p57 is both functionally independent as a cyclin A‐CDK2 inhibitor and unstructured, as demonstrated by circular dichroism and fluorescence spectra indicative of unfolded proteins, a lack of 1H chemical shift dispersion and a hydrodynamic radius consistent with a highly unfolded structure. The amino acid compositions of full‐length p57 and the excised QT domain of p57 exhibit significant deviations from the average composition of globular proteins that are consistent with the observed intrinsic disorder. However, the amino acid composition of the CDK inhibition domain of p57 does not exhibit such a striking deviation from the average values observed for proteins, implying that a general low level of hydrophobicity, rather than depletion or enrichment in specific amino acids, contributes to the intrinsic disorder of the excised p57 CDK inhibition domain. Proteins 2002;46:1–7.

Electrostatic contribution of phosphorylation to the stability of the CREB-CBP activator-coactivator complex.

Electrostatic contribution of phosphorylation to the stability of the CREB–CBP activator–coactivator complex

Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Transcriptional activity of the TFIIA four-helix bundle in vivo.

TFIIA contributes to transcription initiation by stabilizing the TBP–TATA interaction and by mediating the response to transcriptional activators and inhibitors. TFIIA contains a six‐stranded β‐sheet domain and a four‐helix bundle. The β‐domain makes functional contacts with DNA and TBP. The role of the four‐helix bundle was investigated using a structure‐based model of this domain (called 4HB). 4HB adopts a highly stable, helical fold, consistent with its structure in the context of TFIIA. Like TBP and other intact transcription factors, 4HB is able to activate transcription in vivo when artificially recruited to a promoter via a heterologous DNA‐binding domain. Thus, in addition to making important contacts with TBP and DNA via the β‐domain, TFIIA makes other specific, functional contacts with the transcriptional machinery via the four‐helix bundle. Proteins 2001;43:227–232.

Insights into activity and inhibition from the crystal structure of human O-GlcNAcase

O-GlcNAc hydrolase (OGA) catalyzes removal of βα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.