Covalently Linking Oligomerization-Impaired GlpF Protomers Does Not Completely Re-establish Wild-Type Channel Activity

Abstract

Integral membrane proteins of the aquaporin family facilitate rapid water flux across cellular membranes in all domains of life. Although the water-conducting pore is clearly defined in an aquaporin monomer, all aquaporins assemble into stable tetramers. In order to investigate the role of protomer–protomer interactions, we analyzed the activity of heterotetramers containing increasing fractions of mutated monomers, which have an impaired oligomerization propensity and activity. In order to enforce interaction between the protomers, we designed and analyzed a genetically fused homotetramer of GlpF, the aquaglyceroporin of the bacterium Escherichia coli (E. coli). However, increasing fractions of the oligomerization-impaired mutant GlpF E43A affected the activity of the GlpF heterotetramer in a nearly linear manner, indicating that the reduced protein activity, caused by the introduced mutations, cannot be fully compensated by simply covalently linking the monomers. Taken together, the results underline the importance of exactly positioned monomer–monomer contacts in an assembled GlpF tetramer.

The hidden intricacies of aquaporins: Remarkable details in a common structural scaffold

“…Together with the strong conservation of the involved residues (Figure 1, Figure S34 ) this highlights the importance of a stable single-file region for AQ(G)P selectivity and permeability. We speculate that the tetrameric arrangement is inevitable to ensure this structural necessity as monomeric Ec AQPZ (196) and Ec GLPF (197–199) exhibited reduced activities in terms of water and ribitol flux, respectively. Monomeric Ec GLPF was also less resistant to proteolysis in E. coli (186), with a significantly stabilized tetrameric assembly in vivo (188).…”

The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold

“…Together with the strong conservation of the involved residues (Figure 1, Figure S34, Supporting Information) this highlights the importance of a stable single‐file region for AQ(G)P selectivity and permeability. We speculate that the tetrameric arrangement is inevitable to ensure this structural necessity as monomeric Ec AQPZ [ 199 ] and Ec GLPF [ 200–202 ] exhibited reduced activities in terms of water and ribitol flux, respectively. Monomeric Ec GLPF was also less resistant to proteolysis in E. coli , [ 189 ] with a significantly stabilized tetrameric assembly in vivo.…”

Prokaryotic Aquaporins

“…Substitution of Trp219 decreases the activity of GlpF and impairs the stability of the tetrameric protein. In addition, a recent study showed that anionic lipids modulate the activity of the aquaglyceroporin GlpF by stabilizing their tetrameric structure [94], which is important for glycerol transport, as increasing fractions of the oligomerization-impaired mutant GlpF E43A affect the activity of the GlpF heterotetramer [95]. AqpZ is also stabilized by various types of lipids, with cardiolipin imparting the most significant resistance to unfolding [96,97].…”