Abstract
Afamin, which is a human blood plasma glycoprotein, a putative multifunctional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and an unusual variability in cell volume and difficulty in solving the structure despite an ∼34% sequence identity with nonglycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite the simplified glycosylation in insect cell-expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilize the orthorhombic crystal form, reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational state of the protein. An iterative strategy using fully automatic experiments available on MASSIF-1 was used to quickly determine the optimal protocol to achieve the phase transition, which should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computational modelling purposes given that the conformational state of the binding sites and the electron density in the binding site, which is likely to result from PEGs, greatly varies between models. This also holds for the analysis of nonspecific low-affinity ligands, where often a variety of fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of gadoteridol molecules is located near the human albumin Sudlow site, and a second gadoteridol molecule is located at an intermolecular site in proximity to domain IA. The data from the co-crystals support modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.
Highlights
Afamin is a human plasma glycoprotein and a member of the albumin gene family that has been reported to be a multifunctional transporter of hydrophobic molecules such as vitamin E (Voegele et al, 2002) and a potential binding partner for Wnt signalling proteins (Mihara et al, 2016)
High afamin concentrations in human plasma are associated with all major parameters for metabolic syndrome, such as high blood glucose, as well as dyslipemia, obesity and high blood pressure, and pre-eclampsia and ovarian cancer (Dieplinger et al, 2009; Kronenberg et al, 2014; Tramontana et al, 2018)
In contrast to human serum albumin, which is exported from the liver as a nonglycosylated chain, human afamin is highly and variably enzymatically glycosylated in vivo (Jerkovic et al, 2005)
Summary
Afamin is a human plasma glycoprotein and a member of the albumin gene family that has been reported to be a multifunctional transporter of hydrophobic molecules such as vitamin E (Voegele et al, 2002) and a potential binding partner for Wnt signalling proteins (Mihara et al, 2016). A reduction of the mole fraction of water surrounding crystals of macromolecules, either by changing the components of the mother liquor or using specific humidity-control devices, can induce phase transitions (Heras & Martin, 2005; Newman, 2006; Russo Krauss et al, 2012) These transitions can lead to an increase in the order within the crystal lattice, resulting in increased diffraction quality or other changes such as an increase in symmetry or reduced diffraction anisotropy (Bowler et al, 2006; Cramer et al, 2000; Hu et al, 2011; Kadlec et al, 2011; Raj et al, 2017; Zerrad et al, 2010; Scherer et al, 2014). The availability of four independent afamin structure models from two different crystal forms provides insight into the flexibility and dynamics of the molecule, which point towards a significant conformational adaptability when binding to hydrophobic molecules, metal chelates or proteins with hydrophobic acylation such as Wnt, supporting the potential function of afamin as a promiscuous transport molecule in human plasma. All crystals were harvested by laser photoablation and cryocooled using the CrystalDirect robot (Zander et al, 2016; Pellegrini et al, 2011)
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