Single hemagglutinin mutations that alter both antigenicity and receptor binding avidity influence influenza virus antigenic clustering.

The hemagglutination inhibition (HAI) assay is the primary measurement used for identifying antigenically novel influenza virus strains. HAI assays measure the amount of reference sera required to prevent virus binding to red blood cells. Receptor binding avidities of viral strains are not usually taken into account when interpreting these assays. Here, we created antigenic maps of human H3N2 viruses that computationally account for variation in viral receptor binding avidities. These new antigenic maps differ qualitatively from conventional antigenic maps based on HAI measurements alone. We experimentally focused on an antigenic cluster associated with a single N145K hemagglutinin (HA) substitution that occurred between 1992 and 1995. Reverse-genetics experiments demonstrated that the N145K HA mutation increases viral receptor binding avidity. Enzyme-linked immunosorbent assays (ELISA) revealed that the N145K HA mutation does not prevent antibody binding; rather, viruses possessing this mutation escape antisera in HAI assays simply by attaching to cells more efficiently. Unexpectedly, we found an asymmetric antigenic effect of the N145K HA mutation. Once H3N2 viruses acquired K145, an epitope involving amino acid 145 became antigenically dominant. Antisera raised against an H3N2 strain possessing K145 had reduced reactivity to H3N2 strains possessing N145. Thus, individual mutations in HA can influence antigenic groupings of strains by altering receptor binding avidity and by changing the dominance of antibody responses. Our results indicate that it will be important to account for variation in viral receptor binding avidity when performing antigenic analyses in order to identify genuine antigenic differences among influenza virus variants.

Selective complexation of K+ and Na+ in simple polarizable ion-ligating systems.

An influx of experimental and theoretical studies of ion transport protein structure has inspired efforts to understand underlying determinants of ionic selectivity. Design principles for selective ion binding can be effectively isolated and interrogated using simplified models composed of a single ion surrounded by a set of ion-ligating molecular species. While quantum mechanical treatments of such systems naturally incorporate electronic degrees of freedom, their computational overhead typically prohibits thorough dynamic sampling of configurational space and, thus, requires approximations when determining ion-selective free energy. As an alternative, we employ dynamical simulations with a polarizable force field to probe the structure and K(+)/Na(+) selectivity in simple models composed of one central K(+)/Na(+) ion surrounded by 0-8 identical model compounds: N-methylacetamide, formamide, or water. In the absence of external restraints, these models represent gas-phase clusters displaying relaxed coordination structures with low coordination number. Such systems display Na(+) selectivity when composed of more than ∼3 organic carbonyl-containing compounds and always display K(+) selectivity when composed of water molecules. Upon imposing restraints that solely enforce specific coordination numbers, we find all models are K(+)-selective when ∼7-8-fold ion coordination is achieved. However, when models composed of the organic compounds provide ∼4-6-fold coordination, they retain their Na(+) selectivity. From these trends, design principles emerge that are of basic importance in the behavior of K(+) channel selectivity filters and suggest a basis not only for K(+) selectivity but also for modulation of block and closure by smaller ions.

K+/Na+ selectivity in toy cation binding site models is determined by the 'host'.

The macroscopic ion-selective behavior of K(+) channels is mediated by a multitude of physiological factors. However, considering the carbonyl-lined binding site of a conductive K(+) channel as a canonical eightfold coordinated construct can be useful in understanding the principles that correlate the channel's structure with its function. We probe the effects of structure and chemical composition on the K(+)/Na(+) selectivity provided by a variety of simplified droplet-like ion binding site models. We find that when carbonyl- and water-based models capture the qualitative structural features of the K(+) channel binding site, a selective preference for K(+) emerges. Thus our findings suggest that the preference for K(+) over Na(+) exhibited by such models is principally built-in, and is not due to a unique K(+)-selective property of carbonyl functional groups. This suggestion is confirmed by a general thermodynamic assessment, which provides a basis for using simplified models to study the design principles underlying the molecular evolution of K(+) channels.

Statistical determinants of selective ionic complexation: ions in solvent, transport proteins, and other "hosts".

To provide utility in understanding the molecular evolution of ion-selective biomembrane channels/transporters, globular proteins, and ionophoric compounds, as well as in guiding their modification and design, we present a statistical mechanical basis for deconstructing the impact of the coordination structure and chemistry of selective multidentate ionic complexes. The deconstruction augments familiar ideas in liquid structure theory to realize the ionic complex as an open ion-ligated system acting under the influence of an "external field" provided by the host (or surrounding medium). Using considerations derived from this basis, we show that selective complexation arises from exploitation of a particular ion's coordination preferences. These preferences derive from a balance of interactions much like that which dictates the Hofmeister effect. By analyzing the coordination-state space of small family IA and VIIA ions in simulated fluid media, we derive domains of coordinated states that confer selectivity for a given ion upon isolating and constraining particular attributes (order parameters) of a complex comprised of a given type of ligand. We demonstrate that such domains may be used to rationalize the ion-coordinated environments provided by selective ionophores and biological ion channels/transporters of known structure, and that they can serve as a means toward deriving rational design principles for ion-selective hosts.

Deprotonation by dehydration: the origin of ammonium sensing in the AmtB channel.

The AmtB channel passively allows the transport of NH4(+) across the membranes of bacteria via a "gas" NH3 intermediate and is related by homology (sequentially, structurally, and functionally) to many forms of Rh protein (both erythroid and nonerythroid) found in animals and humans. New structural information on this channel has inspired computational studies aimed at clarifying various aspects of NH4(+) recruitment and binding in the periplasm, as well as its deprotonation. However, precise mechanisms for these events are still unknown, and, so far, explanations for subsequent NH3 translocation and reprotonation at the cytoplasmic end of the channel have not been rigorously addressed. We employ molecular dynamics simulations and free energy methods on a full AmtB trimer system in membrane and bathed in electrolyte. Combining the potential of mean force for NH4(+)/NH3 translocation with data from thermodynamic integration calculations allows us to find the apparent pKa of NH4(+) as a function of the transport axis. Our calculations reveal the specific sites at which its deprotonation (at the periplasmic end) and reprotonation (at the cytoplasmic end) occurs. Contrary to most hypotheses, which ascribe a proton-accepting role to various periplasmic or luminal residues of the channel, our results suggest that the most plausible proton donor/acceptor at either of these sites is water. Free-energetic analysis not only verifies crystallographically determined binding sites for NH4(+) and NH3 along the transport axis, but also reveals a previously undetermined binding site for NH4(+) at the cytoplasmic end of the channel. Analysis of dynamics and the free energies of all possible loading states for NH3 inside the channel also reveal that hydrophobic pressure and the free-energetic profile provided by the pore lumen drives this species toward the cytoplasm for protonation just before reaching the newly discovered site.

A simple topological representation of protein structure: implications for new, fast, and robust structural classification.

A topological representation of proteins is developed that makes use of two metrics: the Euclidean metric for identifying natural nearest neighboring residues via the Delaunay tessellation in Cartesian space and the distance between residues in sequence space. Using this representation, we introduce a quantitative and computationally inexpensive method for the comparison of protein structural topology. The method ultimately results in a numerical score quantifying the distance between proteins in a heuristically defined topological space. The properties of this scoring scheme are investigated and correlated with the standard Calpha distance root-mean-square deviation measure of protein similarity calculated by rigid body structural alignment. The topological comparison method is shown to have a characteristic dependence on protein conformational differences and secondary structure. This distinctive behavior is also observed in the comparison of proteins within families of structural relatives. The ability of the comparison method to successfully classify proteins into classes, superfamilies, folds, and families that are consistent with standard classification methods, both automated and human-driven, is demonstrated. Furthermore, it is shown that the scoring method allows for a fine-grained classification on the family, protein, and species level that agrees very well with currently established phylogenetic hierarchies. This fine classification is achieved without requiring visual inspection of proteins, sequence analysis, or the use of structural superimposition methods. Implications of the method for a fast, automated, topological hierarchical classification of proteins are discussed.

Immune history shapes specificity of pandemic H1N1 influenza antibody responses.

Human antibody responses against the 2009 pandemic H1N1 (pH1N1) virus are predominantly directed against conserved epitopes in the stalk and receptor-binding domain of the hemagglutinin (HA) protein. This is in stark contrast to pH1N1 antibody responses generated in ferrets, which are focused on the variable Sa antigenic site of HA. Here, we show that most humans born between 1983 and 1996 elicited pH1N1 antibody responses that are directed against an epitope near the HA receptor-binding domain. Importantly, most individuals born before 1983 or after 1996 did not elicit pH1N1 antibodies to this HA epitope. The HAs of most seasonal H1N1 (sH1N1) viruses that circulated between 1983 and 1996 possess a critical K133 amino acid in this HA epitope, whereas this amino acid is either mutated or deleted in most sH1N1 viruses circulating before 1983 or after 1996. We sequentially infected ferrets with a 1991 sH1N1 virus and then a pH1N1 virus. Sera isolated from these animals were directed against the HA epitope involving amino acid K133. These data suggest that the specificity of pH1N1 antibody responses can be shifted to epitopes near the HA receptor-binding domain after sequential infections with sH1N1 and pH1N1 viruses that share homology in this region.

On the equivalence point for ammonium (de)protonation during its transport through the AmtB channel.

Structural characterization of the bacterial channel, AmtB, provides a glimpse of how members of its family might control the protonated state of permeant ammonium to allow for its selective passage across the membrane. In a recent study, we employed a combination of simulation techniques that suggested ammonium is deprotonated and reprotonated near dehydrative phenylalanine landmarks (F107 and F31, respectively) during its passage from the periplasm to the cytoplasm. At these landmarks, ammonium is forced to maintain a critical number ( approximately 3) of hydrogen bonds, suggesting that the channel controls ammonium (de)protonation by controlling its coordination/hydration. In the work presented here, a free energy-based analysis of ammonium hydration in dilute aqueous solution indicates, explicitly, that at biological pH, the transition from ammonium (NH(4)(+)) to ammonia (NH(3)) occurs when these species are constrained to donate three hydrogen bonds or less. This result demonstrates the viability of the proposal that AmtB indirectly controls ammonium (de)protonation by directly controlling its hydration.

Selectivity in K+ channels is due to topological control of the permeant ion's coordinated state.

The selectivity filter of K+ channels provides specific coordinative interactions between dipolar carbonyl ligands, water, and the permeant cation, which allow for selective flow of K+ over (most importantly) Na+ across the cell membrane. Although a structural viewpoint attributes K+ selectivity to coordination geometry provided by the filter, recent molecular dynamics simulation studies attribute it to dynamic and unique chemical/electrostatic properties of the filter's carbonyl ligands. Here we provide a simple theoretical analysis of K+ and Na+ complexation with water in the context of simplified binding site models and bulk solution. Our analysis reveals that water molecules and carbonyl groups can both provide K+ selective environments if equivalent constraints are imposed on the coordination number of the complex. Absence of such constraints annihilates selectivity, demonstrating that whether a coordinating ligand is a water molecule or a carbonyl group, "external" or "topological" constraints/forces must be imposed on an ion-coordinated complex to elicit selective binding. These forces must inevitably originate from the channel protein, because in bulk water, which, by definition, presents a nonselective medium, the coordination number is allowed to relax to suit the ion. We show that the coordination geometry of K+ channel binding sites is replicated by 8-fold complexation of K+ in both water and simplified binding site models due to dominance of local interactions within a complex and is thus a requirement for topologically constraining the coordination number to a specific value.

Exterior site occupancy infers chloride-induced proton gating in a prokaryotic homolog of the ClC chloride channel.

The ClC family of anion channels mediates the efficient, selective permeation of Cl(-) across the biological membranes of living cells under the driving force of an electrochemical gradient. In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl(-) anion. We infer details of this gating mechanism by studying the free energetics of Cl(-) occupancy in the pore of a prokaryotic ClC homolog. These free energetics were gleaned from 30 ns of molecular dynamics simulation on an approximately 133,000-atom system consisting of a hydrated membrane embedded StClC transporter. The binding sites for Cl(-) in the transporter were determined for the cases where the putative gating residue, Glu(148), was protonated and unprotonated. When the glutamate gate is protonated, Cl(-) favorably occupies an exterior site, S(ext), to form a queue of anions in the pore. However, when the glutamate gate is unprotonated, Cl(-) cannot occupy this site nor, consequently, pass through the pore. An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu(148). Although this suggests that, for the prokaryotic homolog, protonation of Glu(148) may be the first step in transporting Cl(-) at the expense of H(+) transport in the opposite direction, an evolutionary argument might suggest that Cl(-) opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation. During an additional 20 ns free dynamics simulation, the newly discovered outermost site, S(out), and the innermost site, S(int), were seen to allow spontaneous exchange of Cl(-) ions with the bulk electrolyte while under depolarization conditions.