Native mass spectrometry approaches to study zinc-binding plasma proteins

Abstract

Human serum albumin (HSA) is a plasma protein that fulfils a wide range of biological functions and is thought to be the major Zn2+ transporter in blood plasma. The high affinity Zn2+ binding site (Site A) has recently been characterised and is located at an interdomain site. In addition to metal binding, HSA is also important in the transport of fatty acids. Previous work has shown that the binding of Zn2+ at Site A and the binding of myristate at fatty acid site 2 are mutually exclusive. It has been predicted that upon fatty acid binding, a conformational change occurs that can disrupt the residues that form Site A. This allosteric interaction could have an impact on the Zn2+ dependent activities of histidine-rich glycoprotein (HRG), a plasma protein involved in blood coagulation. The purpose of this work was to investigate the metal-binding properties of HSA and a peptide derived from HRG using a native MS approach. Furthermore, the possible Zn2+ transfer between the proteins was explored and also whether fatty acids influenced the Zn2+ distribution. Native ESI-MS was able to detect Zn2+ ions associating with HSA although the interactions with fatty acids appeared to be broken upon entering the gas phase. No apparent loss of Zn2+ from HSA was observed by ESI-MS following incubation with myristate which was confirmed by elemental analysis in solution. Travelling wave ion mobility-MS showed no significant conformational changes between apo-HSA and holo-HSA although Zn2+ appears to have a role in stabilising the domain I/II interface. HSA incubated with myristate showed a larger collisional cross section that is in agreement with the X-ray crystal structures. A peptide mimicking the His-rich region of HRG, HRGP330, was found to bind up to 5 Zn2+ ions by ESI-MS and evidence from a combination of circular dichroism spectroscopy, ion mobility and top-down MS/MS indicated that a conformational change occurs upon Zn2+ binding. During CID and ETD, Zn2+-binding fragments were able to be detected in order to map which residues Zn2+ was bound to. However, numerous fragments were detected and so it would appear that several possible binding sites in HRGP330 have a similar binding affinity for Zn2+. Complementary ESIMS and elemental analysis showed that up to 90% of Zn2+ was transferred from HSA to HRGP330 even in the absence of fatty acid. Cu2+ also preferentially bound to HRGP330 over the N-terminal peptide mimic of HSA. Overall this could have implications for how these metal ions are transported in blood plasma as it would appear from this evidence that HRG is a significant competitor for metal ions bound to HSA