Structure-function investigations of human afamin

The glycoprotein afamin is a blood plasma biomarker whose elevated blood levels are associated with all major parameters for the metabolic syndrome such as high blood glucose levels, abnormal levels of blood lipids, obesity, high blood pressure, and preeclampsia (including elevated risk for premature birth). Despite the association of afamin with widely prevalent symptoms and risk factors for life style diseases, only little is known about its function. From studies in our group at the Medical University Innsbruck we understand that afamin is primarily a promiscuous transporter of lipophilic molecules, particularly vitamin E. Practically nothing is known about the detailed molecular structure of this important biomarker and its relationship to function. The objective of our internationally collaborative project focuses on determining the detailed X-ray crystal structure of afamin and its ligand complexes. Its analysis will lead to new discoveries and provide insights into a number of important questions: How does afamin look in detail? How is it different from other, known transport proteins such as albumin? Which potential active binding sites can we identify? Which classes of small molecules might be able to bind? Are any molecules already natively bound to afamin and can we experimentally complex it with potential ligands? Could afamin transport therapeutic drugs as albumin does and thus modify their effectiveness? For X-ray structure determination, small crystals of the protein afamin need to be grown in tiny drops. We have made significant progress in necessary expression, production and purification of multiple recombinant afamin variants in various cell lines, including glycosylation variants, unglycosylated mutants, and antibody-fragments and complexes for crystallization scaffolding. The analysis of X-ray diffraction data from crystals allows determining the detailed molecular structure of afamin. The resulting structure model provides the basis for structure-guided bioinformatics to detect and analyse binding pockets and for ligand docking studies. We have tested these methods against a homology model of afamin based on the albumin structure and identified a primary, tocopherol (vitamin E) binding pocket as well as various potential secondary hydrophobic binding pockets. The actual experimental structure of afamin will allow screening functionally important binding pockets also for their potential to bind therapeutic drugs and direct subsequent experimental verification. The analysis of the molecular structure of afamin and of its complexes with small molecule ligands is an effective means to reveal the origin of its functional diversity, to allow critical evaluation of its transport properties, and to investigate whether and how it might have any potential as a possible therapeutic target. The results from this study will be completely novel and of significant ground breaking interest in the structural biology and biomedical atherosclerosis research community.

Structure-guided investigations of the human glycoprotein afamin reveal its structural plasticity and multifunctionality. The glycoprotein afamin is a blood plasma biomarker whose elevated blood levels are associated with all major parameters for the metabolic syndrome such as high blood glucose levels, abnormal levels of blood lipids, obesity, high blood pressure, and preeclampsia (including elevated risk for premature birth). Despite the association of afamin with widely prevalent symptoms and risk factors for lifestyle diseases, only little was known about its function and nothing was known about its molecular structure. Studies at the Medical University Innsbruck revealed that afamin is primarily a promiscuous transporter of lipophilic molecules. Within this FWF funded project we were able to reveal the details of the afamin molecular structure and the connection to its function. The structural details provided novel insights and new discoveries, answering important questions about afamin's presumed functions and also revealed completely new functional aspects. The determination of the detailed X-ray crystal structure of afamin was exceptionally laborious because afamin turned out to be extraordinarily flexible and thus has very little incentive to form well-ordered crystals which are the indispensable prerequisite for X-ray crystal structure analysis. Hundreds of tiny afamin crystals had to be grown and examined at one of Europe's most powerful X-ray radiation sources, the ESRF synchrotron in Grenoble. Using the most sophisticated and innovative experimental techniques, we could establish highly detailed structure models revealing the binding pockets and bound molecules. Our structural analysis revealed that afamin can assume multiple conformations, enabling it to accommodate poorly soluble hydrophobic molecules in its deep binding pockets. Unexpected was the discovery that afamin can also bind hydrophobic signalling proteins, which play an important role in intercellular communication, and thus pointing towards a causal connection of afamin with the symptoms of metabolic syndrome. In addition, we discovered that afamin can transport gadoteridol, a paramagnetic NMR tomography contrast enhancement medium. Our detailed molecular structure models of afamin are publicly available for all researchers and for the first time allow a critical and rational investigation of afamin's transport properties as well as a structure-based evaluation of its therapeutic potential. The results from our study are completely novel and of significant ground-breaking interest for the structural biology and biomedical research community.