Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein, whereas beta-DG is an integral membrane protein that also interacts with dystrophin in the cytoplasm. alpha-DG's glycosylation is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (a.a 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the alpha-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation state. We have been investigating the structures of N-terminal region of the WT human alpha-DG (WT-ha-DG) and of the two point mutants V72I (V72I-ma-DG) and D109N (D109N-ma-DG) of mouse alpha-DG by X-ray crystallography, and in solution by Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal alpha-DG that are functionally relevant for its glycosylation pathway. The crystal structure of the ha-DG does not significantly deviate from the already determined crystal structure of WT mouse alpha-DG (WT-ma-DG). The overall fold is conserved and differences are restricted to the most flexible part of the protein, i.e. the loop encompassing residues 159-179, which is only partially visible after crystal structure refinement. In addition, the crystal structures of the two mutants V72I-ma-DG and D109N-ma-DG display the same overall structure of WT-ma-DG, suggesting for negligible effects of the point mutations on the overall fold of alpha-DG. The two mutants show limited and local structural dissimilarities with respect to WT-ma-DG, probably influencing the interaction with potential binding partners. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation, with the most populated close to the crystal conformation and a second less populated one, which is more extended with respect to the principal conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.
Insights into the structure of the N-terminal region of alpha-dystroglycan: a concerted crystallographic and SAXS study
Alberto Cassetta;Sonia Covaceuszach;Francesca Sciandra;Andrea Brancaccio
2016
Abstract
Dystroglycan (DG) is a glycoprotein complex that links the cytoskeleton with the extracellular matrix. DG is composed of two subunits: alpha-DG and beta-DG. alpha-DG is a highly glycosylated extracellular protein, whereas beta-DG is an integral membrane protein that also interacts with dystrophin in the cytoplasm. alpha-DG's glycosylation is essential for high-affinity binding of extracellular matrix proteins such as laminins. The hypoglycosylation of alpha-DG weakens this interaction affinity resulting in severe pathological states. The N-terminal region (a.a 50-313 in mouse) of alpha-DG plays a crucial role in the glycosylation of the alpha-DG mucin-like domain, being required by the glycosyltransferase LARGE during the extension of the O-glycans implicated in laminin binding. Furthermore, pathological missense mutations, observed at the N-terminal region of alpha-DG, are responsible of alpha-DG hypoglycosylation state. We have been investigating the structures of N-terminal region of the WT human alpha-DG (WT-ha-DG) and of the two point mutants V72I (V72I-ma-DG) and D109N (D109N-ma-DG) of mouse alpha-DG by X-ray crystallography, and in solution by Small Angle X-ray Scattering (SAXS). The purpose of this study is to gain evidences about the structural determinants of N-terminal alpha-DG that are functionally relevant for its glycosylation pathway. The crystal structure of the ha-DG does not significantly deviate from the already determined crystal structure of WT mouse alpha-DG (WT-ma-DG). The overall fold is conserved and differences are restricted to the most flexible part of the protein, i.e. the loop encompassing residues 159-179, which is only partially visible after crystal structure refinement. In addition, the crystal structures of the two mutants V72I-ma-DG and D109N-ma-DG display the same overall structure of WT-ma-DG, suggesting for negligible effects of the point mutations on the overall fold of alpha-DG. The two mutants show limited and local structural dissimilarities with respect to WT-ma-DG, probably influencing the interaction with potential binding partners. In contrast, the solution structural models obtained by SAXS analysis depict a different scenario, where the WT-a-DG is quite flexible in solution, assuming more than one conformation, with the most populated close to the crystal conformation and a second less populated one, which is more extended with respect to the principal conformation. The comparison of SAXS data from WT-ma-DG and its mutants demonstrates the presence of a perturbation, in both the conformations as well as in the partition among different populations. SAXS data thus suggest a more complex and dynamical situation with respect to the crystallographic evidences that may have functional implications for DG post-translational processing and for the interactions with its binding partners.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
