Effect of Fc-Glycan Structure on the Conformational Stability of IgG Revealed by Hydrogen/Deuterium Exchange and Limited Proteolysis

Biochemistry ◽  
2016 ◽  
Vol 55 (6) ◽  
pp. 860-868 ◽  
Author(s):  
Jing Fang ◽  
Jason Richardson ◽  
Zhimei Du ◽  
Zhongqi Zhang
Keyword(s):  
Conformational Stability ◽  
Limited Proteolysis ◽  
Deuterium Exchange ◽  
Glycan Structure ◽  
Hydrogen Deuterium Exchange ◽  
Hydrogen Deuterium

scholarly journals Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange

2010 ◽  
Vol 84 (19) ◽  
pp. 10311-10321 ◽  
Author(s):  
Leopold Kong ◽  
Chih-chin Huang ◽  
Stephen J. Coales ◽  
Kathleen S. Molnar ◽  
Jeff Skinner ◽  
...  
Keyword(s):  
Bound States ◽  
Conformational Stability ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Amide Hydrogen ◽  
Binding Reaction ◽  
Binding Surface ◽  
Outer Domain ◽  
Hydrogen Deuterium ◽  
Hiv 1

ABSTRACT The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.

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