Implications of molecular interactions for protein structure, function and design

Abstract

Protein structures are kept in a delicate balance of stability by the interactions of the amino acid residues among themselves, the solvent and other molecules. On one hand they must be stable enough not to unfold, on the other hand they must be mobile enough to undergo structural changes if necessary. Only thus they are able to fulfil their various functions in living organisms, e.g. the catalytic function of an enzyme, protein-ligand recognition or the rapid reorganisation of the cytoskeleton. In this work, we have investigated the contributions of such molecular interactions to protein structure in a functional enzyme, cystathionine [beta]-synthase. We have further analysed the contribution of ionic interactions to the stability of various designed peptides which form coiled coils. Finally, we have collected a statistics of electrostatic interactions in naturally occurring coiled coils to find out which ionic interactions significantly contribute to a successful formation of coiled coils. The results have important implications for the design of coiled-coil proteins. Cystathionine [beta]-synthase is an enzyme of the transsulfuration pathway in eukaryotic cells which catalyses the condensation of serine and homocysteine to yield cystathionine in a pyridoxal 5'-phosphate-dependent [beta]-replacement reaction. The human enzyme also contains heme as a second cofactor which is not required for catalysis. We have solved the structure of the catalytic domain of human cystathionine [beta]-synthase by X-ray crystallography. This is the first protein structure containing a heme binding motif where the iron of the heme is coordinated by a histidine and a cysteine residue. We have also discovered an oxidoreductase active site motif on the surface which might play a role in enzyme regulation. There are more than 100 point mutations known in this enzyme which can cause homocysteinurea disease in humans, characterised by dislocated eye lenses, skeletal problems, vascular disease and mental retardation. We have mapped the mutations in the catalytic domain on the structure and were able to find an explanation for the harmful effect of some mutations by analysing the molecular interactions of the concerned residues. Coiled coils are a simple and regular structural motif in proteins consisting of [alpha]-helices which coil around each other. They can form dimers, trimers, tetramers and pentamers depending on their amino acid sequence and the environment. The principles and factors which lead to this specific fold can therefore be studied in detail. The stability of coiled coils is mainly achieved by the systematic packing of the side chains of the residues at the interface between the helices, called knobs-into-holes packing. We could show, however, that a complex network of inter- and intrahelical salt bridges also contributes significantly to coiled-coil stability by designing short peptides which form dimeric or trimeric coiled coils. The importance of the ionic interactions could be demonstrated by removing a single interhelical salt bridge which abolished the formation of the coiled coil. The peptides were characterised by circular dichroism, analytical ultracentrifugation and X-ray crystallography. We have developed the computer program SBSCC to collect a statistics of intrahelical salt bridges in pure [alpha]-helices and coiled coils from the protein database. We have identified the salt-bridge configurations that have the highest probability to form the ionic interaction and which occur most frequently in [alpha]-helices and coiled coils. We have found interesting differences between [alpha]-helices, parallel and antiparallel 2-stranded coiled-coils with important implications for the coiled-coil design. We have found a positive correlation between the probabilities of different salt-bridge configurations to form the ionic interaction and their frequencies in [alpha]-helices and coiled coils. This indicates that nature relies indeed on ionic interactions to stabilise [alpha]-helices and coiled coils, an issue which was hitherto controversially discussed.