Engineering of a synthetic antibody fragment for structural and functional studies of K+ channels.

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.

A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant E71V.

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.

Barium blockade of the KcsA channel in open and closed conformation datasets.

Barium is a potent blocker of the KcsA potassium channel. A strategy using x-ray crystallography and molecular dynamics (MD) simulation has been used to understand this phenomenon as described in Rohaim et al. [1]. Wild type KcsA is purified to homogeneity and crystallized in low and high K+ conditions. Crystals are grown using the hanging drop vapor diffusion method. To examine barium binding in the selectivity filter of KcsA, the crystals are systemically soaked in various concentrations of barium chloride solution. X-ray crystallography datasets are collected at the Advanced Photon Source. A total of 10 datasets are collected for various barium ion concentrations. Diffraction data are processed using the crystallography pipeline software RAPID. The crystal structures are solved by molecular replacement methods. The structure models are visualized using COOT and refined using REFMAC. Anomalous map coefficients are calculated using the phenix.maps tool in the PHENIX software suite. The datasets are deposited in the Protein Data Bank. The data provides a detailed picture of barium ion interaction with potassium channels. Structural analysis of the KcsA channel reveals two distinct configurations, open- and closed- state. Further MD simulation analysis suggests an energetically favorable binding mechanism for barium ion in the selectivity filter. The data could be used to interpret functional experiments related to barium blockade for potassium channels. Also, it is valuable for comparison and cross validation with other relevant potassium channel structures.

Open and Closed Structures of a Barium-Blocked Potassium Channel.

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.

Glycine substitution in SH3-SH2 connector of Hck tyrosine kinase causes population shift from assembled to disassembled state.

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.

1H, 15N, and 13C resonance assignments of the intrinsically disordered SH4 and Unique domains of Hck.

Hematopoietic 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 eight 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 1H, 15N and 13C chemical shifts of the Hck SH4-Unique domains at pH 4.5.

Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola.

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.

A structural study of ion permeation in OmpF porin from anomalous X-ray diffraction and molecular dynamics simulations.

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.

A conformational intermediate in glutamate receptor activation.

Ionotropic 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.

Multidomain assembled states of Hck tyrosine kinase in solution.

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.

Cation-selective pathway of OmpF porin revealed by anomalous X-ray diffraction.

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.

Atomic constraints between the voltage sensor and the pore domain in a voltage-gated K+ channel of known structure.

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.

Characterization of the natural killer T-cell response in an adoptive transfer model of atherosclerosis.

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.

Site-specific influence of polyunsaturated fatty acids on atherosclerosis in immune incompetent LDL receptor deficient mice.

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.

Autoantibodies to OxLDL fail to alter the clearance of injected OxLDL in apolipoprotein E-deficient mice.

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.

Genetic background selectively influences innominate artery atherosclerosis: immune system deficiency as a probe.

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 approximately 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 approximately 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.