J.E. Pitts

Adaptation of plasminogen activator sequences to known protease structures

The sequences of urokinase (UK) and tissue‐type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their ‘structurally conserved regions’. In spite of its trypsin‐like specificity UK was model‐built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

Calculated tyrosyl circular dichroism of proteins: Absence of tryptophan and cystine interferences in avian pancreatic polypeptide

CALCULATED TYROSYL CIRCULAR DICHROISM OF PROTEINS Absence of tryptophan and cystine interferences in avian pancreatic polypeptide W. STRASSBURGER, U. GLATTER, A. WOLLMER*, J. FLEISCHHAUER., D. A. MERCOLAT T. L. BLUNDELL+, I. GLOVER+, J. E. PITT@, I. J. TICKLE+ and S. P. WOOD’

The crystal structures of three non-pancreatic human insulins

SummaryX-ray studies on semi-synthetic human insulin have shown that it crystallizes in the rhombohedral space group R3 and is nearly isomorphous with 2 Zn pig insulin. Precession photographs of crystals of human and pig insulins show observable changes in the intensity patterns. Crystallographic analysis and refinement of semi-synthetic human insulin at 1.9 Å resolution have shown that its molecular structure is very like that of pig insulin except at the C-terminus of the B chain where the change in sequence occurs. We also report the results of a high resolution crystallographic study of human insulins from different origins. The X-ray diffraction patterns of three non-pancreatic human insulins are indistinguishable from each other and from pancreatic human insulin. Refinement of the structures of the non-pancreatic human insulins has shown that they are identical within the limits of experimental error.

A complete, multi-level conformational clustering of antibody complementarity-determining regions

Classification of antibody complementarity-determining region (CDR) conformations is an important step that drives antibody modelling and engineering, prediction from sequence, directed mutagenesis and induced-fit studies, and allows inferences on sequence-to-structure relations. Most of the previous work performed conformational clustering on a reduced set of structures or after application of various structure pre-filtering criteria. In this study, it was judged that a clustering of every available CDR conformation would produce a complete and redundant repertoire, increase the number of sequence examples and allow better decisions on structure validity in the future. In order to cope with the potential increase in data noise, a first-level statistical clustering was performed using structure superposition Root-Mean-Square Deviation (RMSD) as a distance-criterion, coupled with second- and third-level clustering that employed Ramachandran regions for a deeper qualitative classification. The classification of a total of 12,712 CDR conformations is thus presented, along with rich annotation and cluster descriptions, and the results are compared to previous major studies. The present repertoire has procured an improved image of our current CDR Knowledge-Base, with a novel nesting of conformational sensitivity and specificity that can serve as a systematic framework for improved prediction from sequence as well as a number of future studies that would aid in knowledge-based antibody engineering such as humanisation.

The Three-Dimensional X-Ray Crystal Structure of the Aspartic Proteinase Native to Trichoderma Reesei Complexed with a Renin Inhibitor CP-80794

Aspartic proteinases comprise a family of proteolytic enzymes found in a diverse range of species, from vertebrates, to lower eukaryotes and retroviruses. They are active at neutral or low pH. They are all characterised by the presence of two aspartic acid residues at the active site and are inhibited by pepstatin A. The aspartic proteinases can be broadly divided into two main groups: the retroviral and pepsin-like enzymes. We shall, however only consider the pepsin-like aspartic proteinases.

Protein Engineering Aspartic Proteinases

Aspartic proteinases (1) are presently used in commercial milk, soya and cocoa processing, but there may be other applications in the longer term for designed biocatalysts for the food industry if the thermal stability, pH dependence, size and specificity can be engineered. With this objective in mind we have carried out protein engineering programmes to understand the roles of both individual amino acids and those of loops in aspartic proteinases. We have used chymosin as our model system.

Anisotropic thermal motion and polypeptide secondary structure studied by X-ray analysis at 0.98Å resolution

aPP is a 36-amino acid polypeptide which forms a stable globular structure stabilised by hydrophobic interactions between a polyproline-like helix and an alpha-helix. Crystals contain dimers and are crosslinked by coordination through zinc ions leading to a well-ordered lattice which diffracts X-rays to a resolution of 0.98A. This gives a 5:1 ratio of observations-to-parameters even when anisotropic thermal ellipsoids defined by six parameters for each non-hydrogen atom were refined using least-squares techniques. Rigid body refinement of groups within the polypeptide was also undertaken. The relationship of the principal axes of individual thermal ellipsoids and the librations of rigid side groups to features of secondary, tertiary, and quaternary structures of aPP and its interactions with water molecules are described.

Disjoint combinations profiling (DCP): a new method for the prediction of antibody CDR conformation from sequence

The accurate prediction of the conformation of Complementarity-Determining Regions (CDRs) is important in modelling antibodies for protein engineering applications. Specifically, the Canonical paradigm has proved successful in predicting the CDR conformation in antibody variable regions. It relies on canonical templates which detail allowed residues at key positions in the variable region framework or in the CDR itself for 5 of the 6 CDRs. While no templates have as yet been defined for the hypervariable CDR-H3, instead, reliable sequence rules have been devised for predicting the base of the CDR-H3 loop. Here a new method termed Disjoint Combinations Profiling (DCP) is presented, which contributes a considerable advance in the prediction of CDR conformations. This novel method is explained and compared with canonical templates and sequence rules in a 3-way blind prediction. DCP achieved 93% accuracy over 951 blind predictions and showed an improvement in cumulative accuracy compared to predictions with canonical templates or sequence rules. In addition to its overall improvement in prediction accuracy, it is suggested that DCP is open to better implementations in the future and that it can improve as more antibody structures are deposited in the databank. In contrast, it is argued that canonical templates and sequence rules may have reached their peak.

Protein Engineering of Surface Loops: Preliminary X-Ray Analysis of the Chy155–165rhi Mutant

The protein engineering programme at Birkbeck seeks to develop generic methods based on a design cycle involving biochemical preparation and characterization, determination and comparative analysis of three-dimensional structures, rule-based design, site-directed mutagenesis and expression of the mutants. Knowledge of the tertiary structure of proteins and the use of molecular modelling techniques provide a powerful approach for the design of novel biomolecules with specifically engineered properties. The aspartic proteinase family have been studied in detail by X-ray analysis and provide a suitable data base for the development of protein engineering design principles1–6. This family of endopeptidases belonging to a wide range of biological species with varying substrate specificities. The members of the family are of considerable commercial importance since enzymes such as chymosin and Mucor pusillus pepsin have been exploited by the food industry in cheese and soya processing, while renins, cathepsins and the retroviral proteinases are prime targets of the pharmaceutical industries in drug design.

X-Ray Analysis of Polypeptide Hormones at ≦1Å Resolution: Anisotropic Thermal Motion and Secondary Structure of Pancreatic Polypeptide and Deamino-Oxytocin

Polypeptide hormones are synthesised as larger precursors and stored in endocrine cells from which they are released into the circulation in response to peptide factors, metabolite levels and other agents. At target tissues they bind specific cell surface receptors which give rise to intracellular secondary effects such as changes in the levels of cAMP, tyrosine phosphorylation, etc. Their degradation is rapid and is often mediated by internalisation of the hormone-receptor complex. The role of the conformation of polypeptide hormones during this complex series of events may help in our understanding of the various processes at the molecular level.