The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K þ or Na þ efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K þ to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH in ) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH in first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH in is achieved.antiporter | liposomes | M2 channel | protons | ion channel T he influenza A virus M2 protein is a tetrameric integral membrane protein containing a short N-terminal extracellular domain, a transmembrane (TM) helix, and a 54-residue cytoplasmic tail (1, 2). Four M2 TM domains associate into a highly selective proton channel (3, 4) whose activity is essential for viral replication (5, 6). Influenza virus enters cells via the endosomal pathway, and the M2 protein functions to equilibrate the pH of the virus interior with that of the acidic endosome. The lowering of the pH within the virus leads to disruption of the interactions between the viral ribonucleoprotein (RNP) complex and the M1 protein (5), an important step in viral uncoating (7). Additionally, for some subtypes of influenza A virus, the M2 proton channel activity helps maintain a neutral pH in the lumen of the trans-Golgi network to prevent premature triggering of the hemagglutinin to the low-pH form (8-11). A long-standing question has been how M2 can conduct a substantial number of protons into the very small-volume interior of the virus without developing a substantial electrical gradient. Diffusion of a few protons down their concentration gradient from the acid environment of the endosome into the viral interior would result in an electrical gradient that would abruptly halt further proton flow and thereby prevent acidification of the viral interior. Here we...
Structure-based protein design offers a possibility of optimizing the overall shape of engineered binding scaffolds to match their targets better. We developed a computational approach for the structure-based design of repeat proteins that allows for adjustment of geometrical features like length, curvature, and helical twist. By combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeats (LRRs) from the ribonuclease inhibitor (RI) family that assemble into structures with a predefined geometry. The repeat proteins were built from self-compatible LRRs that are designed to interact to form highly curved and planar assemblies. We validated the geometrical design approach by engineering a ring structure constructed from 10 self-compatible repeats. Protein design can also be used to increase our structural understanding of repeat proteins. We use our design constructs to demonstrate that buried Cys play a central role for stability and folding cooperativity in RI-type LRR proteins. The computational procedure presented here may be used to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired.binding scaffold | Rosetta | buried cysteines | computational protein design | geometrical design
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