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Engineered antibody fragments (Fabs) have made major impacts on structural biology research, particularly to aid structural determination of membrane proteins. Nonetheless, Fabs generated by traditional monoclonal technology suffer from challenges of routine production and storage. Starting from the known IgG paratopes of an antibody that binds to the “turret loop” of the KcsA K+ channel, we engineered a synthetic Fab (sFab) based upon the highly stable Herceptin Fab scaffold, which can be recombinantly expressed in Escherichia coli and purified with single-step affinity chromatography. This synthetic Fab was used as a crystallization chaperone to obtain crystals of the KcsA channel that diffracted to a resolution comparable to that from the parent Fab. Furthermore, we show that the turret loop can be grafted into the unrelated voltage-gated Kv1.2–Kv2.1 channel and still strongly bind the engineered sFab, in support of the loop grafting strategy. Macroscopic electrophysiology recordings show that the sFab affects the activation and conductance of the chimeric voltage-gated channel. These results suggest that straightforward engineering of antibodies using recombinant formats can facilitate the rapid and scalable production of Fabs as structural biology tools and functional probes. The impact of this approach is expanded significantly based on the potential portability of the turret loop to a myriad of other K+ channels.

Barium (Ba2+) is a classic permeant blocker of potassium (K+) channels. The “external lock-in effect” in barium block experiments, whereby the binding of external K+ impedes the forward translocation of the blocker, provides a powerful avenue to investigate the selectivity of the binding sites along the pore of potassium channels. Barium block experiments show that the external lock-in site is highly selective for K+ over Na+. Wild-type KcsA was crystallized in low K+ conditions, and the crystals were soaked in solutions containing various concentrations of barium. Structural analysis reveals open and closed gate conformations of the KcsA channel. Anomalous diffraction experiments show that Ba2+ primarily binds to the innermost site S4 of the selectivity filter of the open-gate conformation and also the site S2, but no binding is detected with the closed-gate conformation. Alchemical free-energy perturbation calculations indicate that the presence of a Ba2+ ion in the selectivity filter boosts the specificity of K+ binding relative to Na+ in the external sites S0–S2.

A large class of K+ channels display a time-dependent phenomenon called C-type inactivation whereby prolonged activation by an external stimulus leads to a non-conductive conformation of the selectivity filter. C-type inactivation is of great physiological importance particularly in voltage-activated K+ channels (Kv), affecting the firing patterns of neurons and shaping cardiac action potentials. While understanding the molecular basis of inactivation has a direct impact on human health, its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the pH-activated bacterial K+ channel KcsA, whose selectivity filter under inactivating conditions adopts a constricted conformation at the level of the central glycine (TTVGYGD) that is stabilized by tightly bound water molecules. However, C-type inactivation is highly sensitive to the molecular environment surrounding the selectivity filter in the pore domain, which is different in Kv channels than in the model KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is consistently substituted by a nonpolar valine in most Kv channels, suggesting that this side chain is an important molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and molecular dynamics simulations of the E71V mutant of KcsA is undertaken to explore the features associated with this Kv-like construct. In both X-ray and ssNMR data, it is observed that the filter of the Kv-like KcsA mutant does not adopt the familiar constricted conformation under inactivating conditions. Rather, the filter appears to adopt a conformation that is slightly narrowed and rigidified over its entire length. No structural inactivation water molecules are present. On the other hand, molecular dynamics simulations indicate that the familiar constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like E71V mutation in the KcsA channel may be associated with different modes of C-type inactivation, showing that distinct selectivity filter environments entail distinct C-type inactivation mechanisms.

C-type inactivation is of great physiological importance in voltage-activated K+ channels (Kv), but its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the bacterial K+ channel KcsA, whose selectivity filter constricts under inactivating conditions. However, the filter is highly sensitive to its molecular environment, which is different in Kv channels than in KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is substituted by a valine conserved in most Kv channels, suggesting that this side chain is a molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and MD simulations of the E71V KcsA mutant is undertaken to explore inactivation in this Kv-like construct. X-ray and ssNMR data show that the filter of the Kv-like mutant does not constrict under inactivating conditions. Rather, the filter adopts a conformation that is slightly narrowed and rigidified. On the other hand, MD simulations indicate that the constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like KcsA mutant may be associated with different modes of C-type inactivation, showing that distinct filter environments entail distinct C-type inactivation mechanisms.

Hemopoietic cell kinase (Hck) is an important signaling enzyme and a potential drug target for HIV infections and Bcr/Abl-chronic myeloid leukemia. The protein shares the same SH4-Unique-SH3-SH2-kinase multi-domain architecture as the other 8 members of the Src family of non-receptor tyrosine kinases. These enzymes are often found anchored to the intracellular side of the membrane via lipidation of the SH4 domain and are integral components of signaling cascades localized at the cell surface. Despite the detailed structural information available for the SH3, SH2, and kinase domains of Hck, the intrinsically disordered nature of the SH4 and Unique domains has resulted in a lack of information for this important region of the protein that is responsible for membrane association. Here, we report the (1)H, (15)N and (13)C chemical shifts of the Hck SH4-Unique domains at pH 4.5.

A combination of small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations based on a coarse grained model is used to examine the effect of glycine substitutions in the short connector between the SH3 and SH2 domains of Hck, a member of the Src-family kinases. It has been shown previously that the activity of cSrc kinase is upregulated by substitution of 3 residues by glycine in the SH3-SH2 connector. Here, analysis of SAXS data indicates that the population of Hck in the disassembled state increases from 25% in the wild type kinase to 76% in the glycine mutant. This is consistent with the results of free energy perturbation calculations showing that the mutation in the connector shifts the equilibrium from the assembled to the disassembled state. This study supports the notion that the SH3-SH2 connector helps to regulate the activity of tyrosine kinases by shifting the population of the active state of the multidomain protein independent of C-terminal phosphorylation.

The Src family kinases (SFKs) are a group of nine non-receptor tyrosine kinases known for their involvement in critical signal transduction pathways. Structurally, SFKs share a common multi-domain architecture, consisting of SH3, SH2, and catalytic (SH1) domains attached to a membrane-anchoring SH4 domain through an intrinsically disordered ∼80 residue region of low sequence conservation called the Unique (U) domain. Despite the wealth of information on SFK structure and function, little is known about the nature of these enzymes in the membrane-associated form. Interestingly, SH4-U domains in different species share common sequence features, despite differences between SFK members, arguing for their importance in differentiating the functions of SFKs and raising questions about their ability to participate in SFK signaling.Hematopoetic cell kinase (Hck), a phagocyte specific proto-oncogene member of the SFKs, is a potential drug target for HIV infections and Bcr/Abl-chronic myeloid leukemia. To achieve structural characterization of the membrane-associated Hck via its SH4-U domains, data from solution and solid-state NMR, neutron reflection, and fluorescence resonance energy transfer were computationally integrated using a novel maximum-entropy multi-resolution “restrained-ensemble molecular dynamics” (reMD) simulation scheme. This framework has allowed us to detail features of the membrane-protein complex at atomic resolution and identify possible new constraints on Hck's ability to phosphorylate downstream targets. In vivo assays are being developed to test the relevance of these findings.

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Src-family kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.

In voltage-gated K(+) channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn(2+) or Cd(2+) with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by approximately 7-8 A and rotated counterclockwise by 37 degrees along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.

Natural killer T (NKT) cells have recently been implicated in atherogenesis, primarily for their ability to recognize and respond to lipid antigens. Because the atherosclerotic lesion is characterized by the retention and modification of lipids in the vascular wall, NKT cells may be involved in promoting the local vascular inflammatory response. Here, we investigate the proatherogenic role of NKT cells in an adoptive transfer model of atherosclerosis, using as recipients immune-deficient, atherosclerosis-susceptible RAG1(-/-)LDLR(-/-) mice. The adoptive transfer of an NKT cell-enriched splenocyte population from Valpha14Jalpha18 T-cell receptor transgenic mice resulted in a 73% increase in aortic root lesion area compared with recipients of NKT cell-deficient splenocytes derived from CD1d(-/-) mice after 12 weeks of Western-type diet feeding. The total serum from hypercholesterolemic mice leads to a small but significant activation of Valpha14Jalpha18 T-cell receptor-expressing hybridoma line by dendritic cells that is CD1d-dependent. Therefore, these studies demonstrate that NKT cells are proatherogenic in the absence of exogenous stimulation, and this activity is likely associated with endogenous lipid antigens carried by lipoproteins in the circulation and perhaps also in the atherosclerotic plaque.

This study tests the hypothesis that autoantibodies to oxidation epitopes on oxidized LDL (OxLDL) promote the clearance of OxLDL from the plasma. Human LDL (hLDL) was injected into immune-competent apolipoprotein E-deficient (apoE(-/-)) mice and immune-deficient apoE(-/-)/recombination-activating gene-deficient mice that lack mature T and B cells and thus antibodies. There was a progressive decrease in human apoB-100 in the plasma in all mice, but the rate of clearance was not greater in the immune-competent mice than in the immune-deficient mice. Interestingly, oxidized phospholipid (OxPL) epitopes as detected by the EO6 antibody on the hLDL increased over time, suggesting de novo oxidation of the LDL or transfer of OxPL to the particles. Because the native LDL was not extensively modified, we also examined the clearance of copper OxLDL. Although the extensively OxLDL was cleared faster than the native LDL, there was no difference in the rate of clearance as a function of immune status. There appeared to be some transfer of OxPL to the endogenous murine LDL. Together, these results suggest that oxidation-specific antibodies do not participate to any great extent in the clearance of OxLDL from plasma. However, it is possible that such antibodies may bind to oxidation epitopes and modulate lesion formation within the vessel wall.

Objective— We sought to examine whether there is a site-specific effect on atherosclerosis of the absence of mature T and B cells caused by a recombination activating-gene deficiency in LDL receptor-deficient mice and whether this effect is influence by the extent of backcrossing to C57BL/6 mice. Methods and Results— Male mice were fed atherogenic diets for 3 months. In strain 1 mice, in which ≈93% of the genes were from C57BL/6 mice, the absence of mature T and B cells led to a significant reduction in atherosclerosis in both the aortic sinus and the innominate artery. In strain 2 mice, in which ≈99+% of the genes were from C57BL/6 mice, immune system deficiency led to a site-specific effect on atherosclerosis, with a reduction in atherosclerosis in the aortic sinus but not in the innominate artery, similar to previous results obtained with apolipoprotein E−/− mice. All of the immune system-incompetent mice had lower plasma total and VLDL cholesterol levels regardless of strain or diet, indicating that differences in lipid levels were unlikely to be responsible for these site-specific effects of immune system deficiency. Conclusions— These results suggest that immune system deficiency has a site-specific effect on atherosclerosis that is sensitive to the genetic background of the mice.

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel., Tetrameric cationic channels specificity is determined by the sequence and structural conformation of their selectivity filter. Here, the authors show that a cationic channel from Tsukamurella paurometabola is non-selective due to a Ca2+-binding motif within its unusual proline-rich filter.

To determine whether T cells and B cells influence lipid metabolism and atherosclerosis, we crossed apolipoprotein E-deficient (apoE degrees ) mice with recombination activating gene 2-deficient (RAG2 degrees ) mice. Total plasma cholesterol levels were approximately 20% higher in male apoE degrees mice compared with the apoE degrees RAG2 degrees mice at 8 weeks of age, and plasma triglyceride levels were 2.5-fold higher in the apoE degrees mice even when plasma cholesterol levels were similar. Male mice with plasma cholesterol levels between 400 and 600 mg/dL at 8 weeks of age were euthanized at 27 and 40 weeks of age. The aortic root lesion area in the apoE degrees RAG2 degrees mice, compared with that in the immune-competent apoE degrees mice, was 81% and 57% smaller at 27 and 40 weeks of age, respectively. In contrast, there was no difference in the size of the brachiocephalic trunk lesions. Similar results were obtained with mice euthanized at 40 weeks of age that had 8-week cholesterol levels between 300 and 399 mg/dL. In apoE degrees RAG2 degrees mice, aortic root atherosclerosis was more profoundly suppressed at lower cholesterol levels. Thus, T and B cells and their products differentially influence the development of atherosclerosis at different sites. We also demonstrate a profound effect of the immune system on plasma lipid homeostasis.

To examine the association of apolipoprotein (apo) E with nascent hepatic lipoproteins we have prepared stable transfectants of the rat hepatoma cell line McA RH7777 expressing the human apoE3 cDNA. When the na- scent lipoproteins secreted from control cells were sepa- rated on fast protein liquid chromatography (FPLC) col- umns, rat apoE was detected in the very low density (VLDL) and high density lipoprotein (HDL) fractions, while rat apoA-I was found in the HDL and lipoprotein free frac- tions. Human apoE was also associated with the VLDL and HDL particles secreted from the transfected McA RH7777 cells. Expression of human apoE resulted in a significant decrease in the amount of rat apoA-I associated with the lip- oprotein particles. Rat apoE was also displaced, but to a lesser extent. Infection of McA RH7777 cells at different multiplicities of infection with recombinant adenoviral vec- tor containing the human apoE cDNA indicated that rat apoA-I was decreased in the HDL fractions at lower levels of expression of human apoE than was rat apoE. The HDL particles were further examined by immunoblotting of non- denaturing gradient gels and by non-denaturing immuno- precipitation. The results indicate that the high density lipoprotein (HDL) particles are heterogeneous in size and apolipoprotein composition with the majority of the rat and human apolipoproteins being located on different particles. These results suggest that the profile and concentration of HDL apolipoproteins produced in hepatocytes influences the assembly of the various subsets of secreted HDL.— Reardon, C. A., L. Blachowicz, K. M. Watson, E. Barr, and G. S. Getz. Association of human apolipoprotein E with lip- oproteins secreted by transfected McA RH7777 cells. J. Lipid Res. 1998. 39: 1372-1381.

OmpF, a multiionic porin from Escherichia coli, is a useful protypical model system for addressing general questions about electrostatic interactions in the confinement of an aqueous molecular pore. Here, favorable anion locations in the OmpF pore were mapped by anomalous X-ray scattering of Br(–) ions from four different crystal structures and compared with Mg(2+) sites and Rb(+) sites from a previous anomalous diffraction study to provide a complete picture of cation and anion transfer paths along the OmpF channel. By comparing structures with various crystallization conditions, we find that anions bind in discrete clusters along the entire length of the OmpF pore, whereas cations find conserved binding sites with the extracellular, surface-exposed loops. Results from molecular dynamics simulations are consistent with the experimental data and help highlight the critical residues that preferentially contact either cations or anions during permeation. Analysis of these results provides new insights into the molecular mechanisms that determine ion selectivity in OmpF porin.

SummaryIonotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an ∼30° rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

The Src tyrosine kinases are large multi-domain enzymes [SH3-SH2-catalytic domain] involved in cellular signaling. Their ability to alternate between catalytically active (high-regulated) and inactive (down-regulated) states in response to specific signals provides a central switching mechanism in cellular transduction pathways. The activity of Src kinases is controlled by the assembly of this multi-domain enzyme. We propose an approach combining small-angle X-ray solution scattering (SAXS) with coarse-grained simulations to characterize quantitatively the multi-domain assembly states of Hck in solution. First, a basis set comprising a small number (∼10) of assembly state “classes” is generated by clustering the configurations obtained from extensive coarse-grained simulations of Hck. Second, the average theoretical SAXS profile for each class of assembly state in the basis set is calculated by using the coarse-grained Fast-SAXS method [Yang et al, Biophys. J. 96:4449 (2009)]. Finally, the relative population of the different classes of assembly states is determined by using a Bayesian-based Monte Carlo procedure seeking to minimize the difference between the theoretical scattering pattern and SAXS data. This novel integrated approach linking experimental SAXS data and simulations is able to resolve the states of assembly of multi-domain Hck in solution under various conditions. The analysis reveals a shift in the equilibrium population of the assembly states upon the binding of various signaling peptides binding to the SH2 or SH3 domains. This integrated approach provides a new way to investigate complex multi-domain assemblies in solution.

The OmpF porin from the Escherichia coli outer membrane folds into a trimer of beta-barrels, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the beta-barrel pore to form a narrow "constriction zone". A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the beta-barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathways for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P6(3) space group and two different data sets were obtained at 2.6 A and 3.0 A resolution with K(+) and Rb(+), respectively. The Rb(+)-soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 A and cation positions were determined. A PEG molecule was observed in the pore region for both the K(+) and Rb(+)-soaked crystals, where it interacts with loop L3. The results reveal the separate pathways of anions and cations across the constriction zone of the OmpF pore.

Src family kinases (SFKs) are a group of nine non-receptor tyrosine kinases that play critical roles in cellular transduction pathways. These highly sought after therapeutic targets are prevalent and promiscuous; therefore, they must be tightly regulated to prevent unchecked signaling cascades. Inter-domain interactions that hinder kinase function are key SFK regulatory mechanisms, and detailed information about these interactions is integral to understanding how these enzymes function.Hck is a SFK primarily found in cells of hematopoietic lineage and is among the most well studied members of the family. Crystal structures of Hck in the down-regulated form and solution studies detailing catalytic responses to regulatory domain displacement provide a robust framework for characterizing the regulatory interactions. Adding to this knowledge, we provide fundamental information on Hck regulation by measuring binding free energies of the native domain-peptide interactions. A combined experimental and computational approach was used to complement and expand the usefulness of the data. The results were compared with bulk FRET measurements made using the full-length kinase to observe how these interactions are modified in the context of the protein.Given the high degree of homology among family members, it is unsurprising that SFKs have some redundant or compensatory functions. However, it has been shown that even the most closely related members of the family cannot always substitute functionally for one another in vivo and the differences responsible have yet to be fully elucidated. Our results lay the groundwork for comparative analysis between different family members and are expected to aid in identifying features that distinguish these enzymes from one another.

Polyunsaturated fatty acids (PUFA) are thought to influence plasma lipid levels, atherosclerosis, and the immune system. In this study, we fed male LDL receptor deficient (LDLR(-/-)) mice and immune incompetent LDLR(-/-) RAG2(-/-) mice diets containing predominantly saturated fats (milk fat) or PUFA (safflower oil) to determine if the response to diet was influenced by immune status. Relative to milk fat diet, plasma lipid and VLDL levels in both the LDLR(-/-) and LDLR(-/-) RAG2(-/-) mice fed safflower oil diet were lower, suggesting that the primary effect of PUFA on plasma lipids was not due to its inhibition of the immune system. Neither diet nor immune status influenced hepatic triglyceride production and post-heparin lipase activity, suggesting that the differences in triglyceride levels are due to differences in rates of catabolism of triglyceride-rich lipoproteins. While both diets promoted atherogenesis, both aortic root and innominate artery atherosclerosis in LDLR(-/-) mice was less in safflower oil fed animals. In contrast, a site-specific effect of PUFA was observed in the immune incompetent LDLR(-/-) RAG2(-/-). In these mice, aortic root atherosclerosis, but not innominate artery atherosclerosis, was less in PUFA fed animal. These results suggest that PUFA and the immune system may influence innominate artery atherosclerosis by some overlapping mechanisms.

The outer membrane of Gram-negative bacteria is a highly specialized structure that lies outside the cytoplasmic membrane and peptidoglycan layer and forms the interface between the cell and its external environment. The outer membrane constitutes a physical and functional barrier between the cell body and its surroundings which effectively controls access of solutes and external agents to the cytoplasmic membrane. The main function of OmpF is to facilitate the translocation of hydrophilic solutes with molecular mass upto 600 Da across the outer membrane. Ion selectivity, the property of porins in distinguishing charged ions is greatly affected by the electrostatics due to the presence of acidic and basic amino acid residues in the pore constriction zone. Detailed experimental and theoretical approaches including MD, BD and continuum electrodiffusion modeling revealed the importance of electrostatic interactions on the transport of cations and anions across the channel. Several computational studies have identified separate pathways for the flow of cations and anions along the OmpF pore. To probe this prediction experimentally, we report here the results from anomalous X-ray scattering data from rubidium and bromide revealing the permeation of cations and anions along the entire length of the OmpF channel.

Outer membrane protein F (OmpF) is a nonspecific, water-filled channel that allows the passage of hydrophilic antibiotics across the lipid barrier of Gram-negative bacteria. The diffusion limit for translocating molecules is provided by the constriction zone, which forms both a steric and an electrostatic hindrance due to a unique arrangement of charged residues that reach into the pore. Antibiotic-resistant bacterial isolates have been found that show either reduced expression of OmpF protein or mutation of key constriction zone residues. Therefore, it is necessary to identify important drug-protein molecular interactions that facilitate efficient transfer across the OmpF channel. We have co-crystallized E. coli OmpF with zwitterionic ampicillin, anionic carbenicillin, and bulky enrofloxacin, and observe the density corresponding to the antibiotic molecules inside the channel. Comparison of the experimental x-ray density with molecular dynamics simulations allows multiple conformations of the diffusing molecule to be resolved. Results of this work give insights into how the charge distribution and size of the translocating molecule affect binding interactions and dynamics within the OmpF constriction zone. We are also using novel computational methods to model transition pathways for various molecules across the entire length of the porin for both WT and mutant OmpF proteins. Together, this work will assist in the design and screening of new antibiotics with improved diffusional characteristics. [Supported by NIH grant GM062342].

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory synaptic transmission in the central nervous system. The free energy of neurotransmitter-binding to the ligand-binding domains (LBDs) of iGluRs is converted into useful work to induce conformational changes that cause receptor activation. Here, the energetic contributions are dissected into the “hidden” components associated with ligand-docking and ligand-induced LBD closure. The relevant thermodynamic contributions have been computed for nine ligands of GluA2 using all-atom molecular dynamics free energy simulations with explicit solvent. The results are compared with experimentally measured apparent affinities to the isolated LBD. An analysis of accessible LBD conformations transposed into the context of an intact GluA2 receptor reveals that the relative displacement of specific diagonal subunits in the tetrameric structure may play a key role in the action of partial agonists. Our X-ray crystal structure of a crosslink-stabilized LBD assembly shows that these diagonal subunits can be covalently linked without disrupting the fold of the LBDs.

Outer membrane protein F (OmpF) allows diffusion of beta-lactam antibiotics across the outer membrane of Gram-negative bacteria. The diffusion limit for translocating molecules is provided by the constriction zone, which defines both the channel diameter at the narrowest region, as well as electrostatic properties due to a unique arrangement of charged residues. Since reduced outer membrane permeability contributes to antimicrobial resistance, it is necessary to identify the role of drug-protein molecular interactions in antibiotic transfer in order to design antibiotics with improved diffusional characteristics. We have co-crystallized E. coli OmpF with various antibiotic molecules and observe the density corresponding to the antibiotic inside the OmpF pore. Results of this work give insights into how the charge distribution of the translocating molecule affects binding interactions within the OmpF constriction zone. Furthermore, functional assays and mutational analysis provide evidence that alteration of some key charged OmpF residues has an effect on bacterial cell survival. We are also using computational methods to model the pathways of diffusing antibiotics and measure their residence time in the OmpF pore. Here we are employing a new approach called the String Method with swarms-of-trajectories to study transition pathways for various zwitterionic and anionic molecules across both WT and mutant OmpF porins. Results of this work will therefore assist in the design of new antibiotics that are more effective in the treatment of bacterial infections. [Supported by NIH grant GM062342].

The ionotropic glutamate receptors (iGluR) mediate the majority of the rapid signalling at excitatory synapses in the brain. The binding of glutamate and other agonist molecules to the ligand-binding domains (LBDs) of the iGluR provides the free energy for driving intra-LBD conformational transitions that open the gate of the ion channel. However, much less information is available about inter-LBD motions. We recently showed that disulfide crosslinks between kainate receptor LBD dimers inhibit receptor activation (Das et al, 2010, PNAS). Here, we used a combination of structural studies and electrophysiology to map the conformational transitions of the LBD dimers between different states of the GluA2 receptor. Interdimer disulfide trapping with exquisite functional sensitivity shows that the two subunit dimers must translate relative to each other during activation, with the center of the dimers moving towards the overall axis of the channel. The crosslink captures an intermediate state between resting and fully activated and has geometry (including reduced linker separation) that provides new insight to glutamate receptor activation.