Understanding of protein-protein interactions provides a means for utilizing nature’s own resources in the development of new and better drugs.
Proteins are the body’s building blocks and are involved in the communication cascades in and between cells. Specific protein-to-protein interactions are involved in the processes, and understanding these interactions provides a means for utilizing nature’s own resources in the development of new and better drugs. Pharmaceutical products derived from proteins are increasingly used as medicine. Examples are insulin and insulin derivatives and recombinant therapeutic proteins such as monoclonal antibodies used for chemo therapy.
Small angle X-ray scattering (SAXS) is a technique which can provide us with information about the shape and behavior of a macromolecule in solution. Where X-ray diffraction gives us detailed atomic resolution from crystals, SAXS is limited in resolution due to tumbling of the molecules. Instead we gain information about the protein in a more native-like environment and the technique is applicable to a wide range of proteins and protein complexes where we are not dependent on the molecule(s) ability to crystallize. At low concentrations SAXS reveals the overall envelope of molecules in solution, while at higher concentrations analysis of the SAXS curve provides information of intermolecular interactions. This means that by using SAXS, it is possible to study both the protein structure and the protein-protein interactions. In this project, SAXS was used to investigate protein structure and protein-protein interactions.
The latter either as specific binding between two proteins to form a protein complex or as non-specific interactions, which refers to the inter-molecular forces that govern a solution and relate to the stability of a protein solution. This has been supplemented by static light scattering and in-silico modelling. The focus has been on the interaction of Human serum albumin (HSA) and target proteins, in particular derivatives of the naturally occurring glucagon-like peptide 1 (GLP-1). HSA is utilized in many ways in the pharmaceutical industry, e.g. in formulations of other proteins and in particular for half-life extension of peptides. GLP-1 is an incretin hormone and derivatives hereof are used in the treatment of diabetes type 2. The very short half-life of this peptide makes the peptide itself unsuitable as a drug but increasing the half-life can be done by methods such as conjugation to other molecules such as HSA.
In order to give a better understanding of the interaction of HSA with other proteins, the behavior of HSA was examined in three different solution systems. The non-specific intermolecular interactions were highly dependent on the other chemicals present in the solution. This is important when investigating binary systems as the self-interaction of HSA could affect the interaction with other proteins. Conjugation of peptides such as GLP-1 to HSA can increase the half-life of the peptide by taking advantage of the large size of HSA and its interaction with the neonatal Fc receptor (FcRn), both of which are responsible for the extraordinary half-life of ~21 days of HSA. It is important that the interactions of the conjugate with the FcRn and the GLP-1 receptor are conserved for optimal retention time and potency. SAXS was used to derive a molecular structure of the conjugate and hereby it was possible to shed light on the molecular mechanism behind the binding studies and the pharmacokinetic studies.
Docking tools can be used to analyze the molecular envelopes derived from SAXS studies on protein complexes, when the structures of the individual proteins are known. This has its limitations though and the last chapter of the thesis presents a theoretical study on the use of the protein docking tool RosettaDock in combination with SAXS. Here it is shown that SAXS decreases the conformational space explored by RosettaDock and ultimately increases the chance of identifying a near native protein complexes.
Illustration:
Crystal structure of HSA in complex with Myristic acids (PDB 1E7G). Domain names are highlighted in bold with FA binding sites in italic. Fatty acids (FA) are presented as grey spheres with oxygens in red. Domain colouring and naming of domains and FA binding sites, have been done in accordance with the nomenclature adopted by Bhattacharya et al.(14). References in the text to the FA binding sites are shorthanded to FA1-7.