The Scottish Structural Proteomics Facility: targets, methods and outputs.

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.

Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding.

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e. the apo-form) has been reported. Here, we describe the solution structure and dynamics of apo-MDM2N and thus reveal the nature of the conformational changes in MDM2N that accompany binding of p53. The new structure suggests that p53 effects displacement of an N-terminal segment of apo-MDM2N that occludes access to the shallow end of the p53-binding cleft. MDM2N must also undergo an expansion upon binding, achieved through a rearrangement of its two pseudosymetrically related sub-domains resulting in outward displacements of the secondary structural elements that comprise the walls and floor of the p53-binding cleft. MDM2N becomes more rigid and stable upon binding p53. Conformational plasticity of the binding cleft of apo-MDM2N could allow the parent protein to bind specifically to several different partners, although, to date, all the known liganded structures of MDM2N are highly similar to one another. The results indicate that the more open conformation of the binding cleft of MDM2N observed in structures of complexes with small molecules and peptides is a more suitable one for ligand discovery and optimisation.

Overexpression, purification, crystallization and data collection of Sulfolobus solfataricus Sso6206, a novel highly conserved protein.

Sso6206, a 10.5 kDa protein from Sulfolobus solfataricus, has been overexpressed, purified and crystallized. The protein crystallizes in space group P6(1/5)22, with unit-cell parameters a = b = 157.8, c = 307.3 A. The crystals are hexagonal bipyramids and a data set has been collected to 2.4 A resolution. Molecular replacement cannot be attempted as no convincing model can be identified. Crystals of selenomethionine-variant protein have not yet been obtained. Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

Insights into cyclin groove recognition: complex crystal structures and inhibitor design through ligand exchange.

Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity. Here we present X-ray structures of the protein complex CDK2/CA, together with the cyclin groove-bound peptides H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH(2) (peptide 1), H-Arg-Arg-Leu-Ile-Phe-NH(2) (peptide 2), Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH(2) (peptide 3), H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH(2) (peptide 4), and H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH(2) (peptide 5). Some of the peptide complexes presented here were obtained through the novel technique of ligand exchange within protein crystals. This method may find general application for obtaining complex structures of proteins with surface-bound ligands.

Does telephone follow-up predict patient satisfaction and readmission?

Nursing telephone calls after hospital discharge are commonly adopted as a tool to improve patient satisfaction and continuity of care. Previous research, however, has been inconclusive on the impact of telephone follow-up. The purpose of this study was to comparatively examine patients who received telephone follow-up for response differences on a mail satisfaction survey and 30-day readmission rates for a large health system in southeast Texas. Telephone follow-up, patient satisfaction, and administrative billing data from 2008 to 2009 were retrospectively examined across 10 nursing units that routinely performed calls after patient discharge. Patients eligible to receive a nursing call (N = 10,559) were categorized based on responses to nursing questions or if no contact was made. Logistic regression was used to evaluate whether call data significantly predicted survey response and 30-day readmission rates. Nonparametric analysis was used to evaluate whether survey ratings varied between groups. Completion of telephone follow-up was a significant (P < 0.01) predictor of patient response to the mail survey, with 62% more patients returning surveys after contact. Completion of a nursing call with a patient who reported a physician appointment was a significant predictor (P = 0.04) of lower 30-day readmissions. Readmission rates were 10.8% for patients who did not receive telephone follow-up compared to 9.5% for patients who received a call and who had a scheduled physician appointment. Mean nursing and overall satisfaction scores varied minimally between groups and telephone follow-up was not a significant predictor of patient satisfaction. Telephone follow-up shows significant predictive value for mail survey response and 30-day readmission rates but does not correlate with patient satisfaction scores in the hospital setting.