Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.     

Membrane association and activity of 15/16-membered peptide antibiotics: Zervamicin IIB, ampullosporin A and antiamoebin I

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K⁺ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER > AMP >> ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC_16:1PC, diC_18:1PC, C_16:0/C_18:1PC, diC_20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.     

Structure of Amantadine-Bound M2 Transmembrane Peptide of Influenza A in Lipid Bilayers from Magic-Angle-Spinning Solid-State NMR: The Role of Ser31 in Amantadine Binding

The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the side-chain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting structural findings from non-bilayer samples.     

NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix-loop-helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [methyl-13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass=75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer-dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB-domain 4 heterodimer. A dissociation constant (KD) value of 3.3 microM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3-4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1-3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB-domain 4 heterodimerization are allowed.     

The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A

Replacement of a cis-proline by glycine at position 114 in ribonuclease A leads to a large decrease in thermal stability and simplifies the refolding kinetics. A crystallographic approach was used to determine whether the decrease in thermal stability results from the presence of a cis glycine peptide bond, or from a localized structural rearrangement caused by the isomerization of the mutated cis 114 peptide bond. The structure was solved at 2.0 A resolution and refined to an R-factor of 19.5% and an R(free) of 21.9%. The overall conformation of the protein was similar to that of wild-type ribonuclease A; however, there was a large localized rearrangement of the mutated loop (residues 110-117-a 9.3 A shift of the Calpha atom of residue 114). The peptide bond before Gly114 is in the trans configuration. Interestingly, a large anomalous difference density was found near residue 114, and was attributed to a bound cesium ion present in the crystallization experiment. The trans isomeric configuration of the peptide bond in the folded state of this mutant is consistent with the refolding kinetics previously reported, and the associated protein conformational change provides an explanation for the decreased thermal stability.     

Macromolecular crowding at membrane interfaces: adsorption and alignment of membrane peptides

Association of proteins to cellular membranes is involved in various biological processes. Various theoretical models have been developed to describe this adsorption mechanism, commonly implying the concept of an ideal solution. However, due to the two-dimensional character of membrane surfaces intermolecular interactions between the adsorbed molecules become important. Therefore previously adsorbed molecules can influence the adsorption behavior of additional protein molecules and their membrane-associated structure. Using the model peptide LAH₄, which upon membrane-adsorption can adopt a transmembrane as well as an in-planar configuration, we carried out a systematic study of the correlation between the peptide concentration in the membrane and the topology of this membrane-associated polypeptide. We could describe the observed binding behavior by establishing a concept, which includes intermolecular interactions in terms of a scaled particle theory.High surface concentration of the peptide shifts the molecules from an in-planar into a transmembrane conformation, a process driven by the reduction of occupied surface area per molecule. In a cellular context, the crowding-dependent alignment might provide a molecular switch for a cell to sense and control its membrane occupancy. Furthermore, crowding might have pronounced effects on biological events, such as the cooperative behavior of antimicrobial peptides and the membrane triggered aggregation of amyloidogenic peptides.     

Role of the membrane interface on the conformation of the caveolin scaffolding domain: a CD and NMR study

Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82-R101 and D82-I109, encompassing the caveolin scaffolding domain (D82-R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82-L109 peptide, mainly located in the V(94)TKYWFYR(101) motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.     

Fusogenic Alzheimer's peptide fragment Abeta (29-42) in interaction with lipid bilayers: secondary structure, dynamics, and specific interaction with phosphatidyl ethanolamine polar heads as revealed by solid-state NMR

The interaction of the native Alzheimer's peptide C-terminal fragment Abeta (29-42), and two mutants (G33A and G37A) with neutral lipid bilayers made of POPC and POPE in a 9:1 molar ratio was investigated by solid-state NMR. This fragment and the lipid composition were selected because they represent the minimum requirement for the fusogenic activity of the Alzheimer's peptide. The chemical shifts of alanine methyl isotropic carbon were determined by MAS NMR, and they clearly demonstrated that the major form of the peptide equilibrated in membrane is not in a helical conformation. (2)H NMR, performed with acyl chain deuterated POPC, demonstrated that there is no perturbation of the acyl chain's dynamics and of the lipid phase transition temperature. (2)H NMR, performed with alanine methyl-deuterated peptide demonstrated that the peptide itself has a limited mobility below and above the lipid phase transition temperature (molecular order parameter equal to 0.94). MAS (31)P NMR revealed a specific interaction with POPE polar head as seen by the enhancement of POPE phosphorus nuclei T(2) relaxation. All these results are in favor of a beta-sheet oligomeric association of the peptide at the bilayer interface, preferentially recruiting phosphatidyl ethanolamine polar heads.     

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala₁₂₂₇ to Ser₁₂₅₁), which contains a single Trp residue (W₁₂₄₆) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala₁₂₂₇ was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring (∼50% α-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp₁₂₄₆ to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including λ_max, lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.     

Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor

Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported; those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.     

Membrane-bound peptides mimicking transmembrane Vph1p helix 7 of yeast V-ATPase: A spectroscopic and polarity mismatch study

The V-ATPases are a family of ATP-dependent proton pumps, involved in a variety of cellular processes, including bone breakdown. V-ATPase enzymes that are too active in the latter process can result in osteoporosis, and inhibitors of the enzyme could be used to treat this disease. As a first step in studying the structure and function of the membrane-embedded interface at which proton translocation takes place, and its role in V-ATPase inhibition, synthetic peptides P1 and P2 consisting of 25 amino acid residues are presented here that mimic Vph1p helix 7 of yeast V-ATPase. A single mutation R10A between peptide P1 and P2 makes it possible to focus on the role of the essential arginine residue R735 in proton translocation. In the present work, we use a novel combination of spectroscopic techniques, such as CD spectroscopy, tryptophan emission spectra, acrylamide quenching and parallax analysis, and polarity mismatch modeling to characterize the peptides P1 and P2 in lipid bilayer systems. Based on both the spectroscopic experiments and the polarity mismatch modeling, P1 and P2 adopt a similar transmembrane conformation, with a mainly α-helical structure in the central part, placing the tryptophan residue at position 12 at a location 4 ± 2 Å from the centre of the lipid bilayer. Furthermore, the arginine at position 10 in P1 does not have an effect on the bilayer topology of the peptide, showing that the long, flexible side chain of this residue is able to snorkel towards the lipid headgroup region. This large flexibility of R735 might be important for its function in proton translocation in the V-ATPase enzyme.     

Regulatory role of sphingomyelin metabolites in hypoxia-induced vascular smooth muscle cell proliferation

The rotating frame nuclear magnetic resonance relaxation rate R(1rho) in the blood and cell lysate was studied at 4.7T to provide reference values for in vivo modeling and to address the mechanisms contributing to net relaxation. A strong dependence on oxygenation, hematocrit, and spin lock field strength B(1) (0.2-1.6G) was observed in whole blood, whereas in lysate the effects were severely attenuated. The results were further compared to transverse relaxation rate R(2). A good agreement in low-field asymptotes of these two relaxation rates was found. R(1rho) field dispersion was fitted to Lorenzian line shape and resulted in correlation times around 40 micros. The dispersion behavior was related to motional properties of intracellular hemoglobin and effects of susceptibility shift interface across the cell membrane induced by compartmentalization of Hb into cells in blood.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Structural Analysis of the Streptomyces avermitilis CYP107W1-Oligomycin A Complex and Role of the Tryptophan 178 Residue

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 Å. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant’s catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.     

Orientation and pore-forming mechanism of a scorpion pore-forming peptide bound to magnetically oriented lipid bilayers

The orientation and pore-forming mechanisms of pandinin 2 (pin2), an antimicrobial peptide isolated from venom of the African scorpion Pandinus imperator, bound to magnetically oriented lipid bilayers were examined by 31P and 13C solid-state, and 15N liquid-state NMR spectroscopy. 31P NMR measurements at various temperatures, under neutral and acidic conditions, showed that membrane lysis occurred only under acidic conditions, and at temperatures below the liquid crystal-gel phase transition of the lipid bilayers, after incubation for two days in the magnet. Differential scanning calorimetry measurements showed that pin2 induced negative curvature strain in lipid bilayers. The 13C chemical shift values of synthetic pin2 labeled at Gly3, Gly8, Leu12, Phe17, or Ser18 under static or slow magic-angle spinning conditions, indicate that pin2 penetrates the membrane with its average helical axis perpendicular to the membrane surface. Furthermore, amide H-D exchange experiments of 15N-Ala4, Gly8, and Ala9 triply-labeled pin2 suggest that this peptide forms oligomers and confirms that the N-terminal region creates membrane pores.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented N and P Solid-State NMR Spectroscopy

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with ¹⁵N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting ¹⁵N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled ³¹P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.     

Phage display and crystallographic analysis reveals potential substrate/binding site interactions in the protein secretion chaperone CsaA from Agrobacterium tumefaciens

The protein CsaA has been proposed to function as a protein secretion chaperone in bacteria that lack the Sec-dependent protein-targeting chaperone SecB. CsaA is a homodimer with two putative substrate-binding pockets, one in each monomer. To test the hypothesis that these cavities are indeed substrate-binding sites able to interact with other polypeptide chains, we selected a peptide that bound to CsaA from a random peptide library displayed on phage. Presented here is the structure of CsaA from Agrobacterium tumefaciens (AtCsaA) solved in the presence and absence of the selected peptide. To promote co-crystallization, the sequence for this peptide was genetically fused to the amino-terminus of AtCsaA. The resulting 1.65 Å resolution crystal structure reveals that the tethered peptide from one AtCsaA molecule binds to the proposed substrate-binding pocket of a symmetry-related molecule possibly mimicking the interaction between a pre-protein substrate and CsaA. The structure shows that the peptide lies in an extended conformation with alanine, proline and glutamine side chains pointing into the binding pocket. The peptide interacts with the atoms of the AtCsaA-binding pocket via seven direct hydrogen bonds. The side chain of a conserved pocket residue, Arg76, has an “up” conformation when the CsaA-binding site is empty and a “down” conformation when the CsaA-binding site is occupied, suggesting that this residue may function to stabilize the peptide in the binding cavity. The presented aggregation assays, phage-display analysis and structural analysis are consistent with AtCsaA being a general chaperone. The properties of the proposed CsaA-binding pocket/peptide interactions are compared to those from other structurally characterized molecular chaperones.     

Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel α1 subunits

The pore-forming component of voltage-gated calcium channels, α₁ subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains I and II of Ca_v1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca_v1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca_v1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly¹⁸² in domain I is involved in the membrane trafficking or surface expression of α₁ subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca_v1.2, although G579Q showed a normal VDI, implying that Gly⁵⁷⁹ in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for α₁ subunit to become functional.