Investigating the conformational landscape of human histidine-rich glycoprotein using amide HDX-MS.

Histidine-rich glycoprotein (HRG) is a multidomain plasma protein involved in immune modulation, angiogenesis, coagulation and fibrinolysis. Despite its broad biological relevance, structural investigations into HRG have yielded only limited information, with no experimentally resolved three-dimensional structures of the intact protein to date. In this study, we integrate hydrogen-deuterium exchange mass spectrometry (HDX-MS) with predictive insights from AlphaFold to map the conformational landscape of HRG in solution under near-native conditions. The N1/N2 domains displayed low solvent exchange overall. However, specific regions of high-solvent exchange were also apparent, providing evidence for more dynamic stretches of secondary structure and the presence of flexible loops within these regions. Our findings also reveal extensive solvent accessibility and rapid exchange kinetics within the histidine-rich region, proline-rich regions and large segments of the C-terminal domain, strongly indicating intrinsic disorder across these domains. These findings support a model in which structural flexibility underlies HRG's capacity to engage with a wide range of molecular partners. This integrative approach offers new insight into the conformational architecture of HRG and lays the groundwork for uncovering the molecular mechanisms governing its biological activity.