Characterization and comparison of two binding sites on obscurin for small ankyrin 1

Obscurin A, an ～720 kDa modular protein of striated muscles, binds to small ankyrin 1 (sAnk1, Ank 1.5), an integral protein of the sarcoplasmic reticulum, through two distinct carboxy-terminal sequences, Obsc(6316-6436) and Obsc(6236-6260). We hypothesized that these sequences differ in affinity but that they compete for the same binding site on sAnk1. We show that the sequence within Obsc(6316-6436) that binds to sAnk1 is limited to residues 6316-6345. Comparison of Obsc(6231-6260) to Obsc(6316-6345) reveals that Obsc(6316-6345) binds sAnk1 with an affinity (133 ± 43 nM) comparable to that of the Obsc(6316-6436) fusion protein, whereas Obsc(6231-6260) binds with lower affinity (384 ± 53 nM). Oligopeptides of each sequence compete for binding with both sites at half-maximal inhibitory concentrations consistent with the affinities measured directly. Five of six site-directed mutants of sAnk1 showed similar reductions in binding to each binding site on obscurin, suggesting that they dock to many of the same residues of sAnk1. Circular dichroism (CD) analysis of the synthetic oligopeptides revealed a 2-fold greater α-helical content in Obsc(6316-6346), ～35%, than Obsc(6231-6260,) ～17%. Using these data, structural prediction algorithms, and homology modeling, we predict that Obsc(6316-6345) contains a bent α-helix of 12 amino acids, flanked by short disordered regions, and that Obsc(6231-6260) has a short, N-terminal α-helix of 4-5 residues followed by a long disordered region. Our results are consistent with a model in which both sequences of obscurin differ significantly in structure but bind to the ankyrin-like repeat motifs of sAnk1 in a similar though not identical manner.     

Hydrophobic residues in small ankyrin 1 participate in binding to obscurin

Abstract Small ankyrin-1 is a splice variant of the ANK1 gene that binds to obscurin A. Previous studies have identified electrostatic interactions that contribute to this interaction. In addition, molecular dynamics (MD) simulations predict four hydrophobic residues in a 'hot spot' on the surface of the ankyrin-like repeats of sAnk1, near the charged residues involved in binding. We used site-directed mutagenesis, blot overlays and surface plasmon resonance assays to study the contribution of the hydrophobic residues, V70, F71, I102 and I103, to two different 30-mers of obscurin that bind sAnk1, Obsc????????? and Obsc?????????. Alanine mutations of each of the hydrophobic residues disrupted binding to the high affinity binding site, Obsc?????????. In contrast, V70A and I102A mutations had no effect on binding to the lower affinity site, Obsc?????????. Alanine mutagenesis of the five hydrophobic residues present in Obsc????????? showed that V6328, I6332, and V6334 were critical to sAnk1 binding. Individual alanine mutants of the six hydrophobic residues of Obsc????????? had no effect on binding to sAnk1, although a triple alanine mutant of residues V6233/I6234/I6235 decreased binding. We also examined a model of the Obsc?????????-sAnk1 complex in MD simulations and found I102 of sAnk1 to be within 2.2? of V6334 of Obsc?????????. In contrast to the I102A mutation, mutating I102 of sAnk1 to other hydrophobic amino acids such as phenylalanine or leucine did not disrupt binding to obscurin. Our results suggest that hydrophobic interactions contribute to the higher affinity of Obsc????????? for sAnk1 and to the dominant role exhibited by this sequence in binding.     

Electrostatic interactions mediate binding of obscurin to small ankyrin 1: biochemical and molecular modeling studies

Small ankyrin 1 (sAnk1; also known as Ank1.5) is an integral protein of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle cells, where it is thought to bind to the C-terminal region of obscurin, a large modular protein that surrounds the contractile apparatus. Using fusion proteins in vitro, in combination with site-directed mutagenesis and surface plasmon resonance measurements, we previously showed that the binding site on sAnk1 for obscurin consists, in part, of six lysine and arginine residues. Here we show that four charged residues in the high-affinity binding site on obscurin for sAnk1 (between residues 6316 and 6345), consisting of three glutamates and a lysine, are necessary, but not sufficient, for this site on obscurin to bind to sAnk1 with high affinity. We also identify specific complementary mutations in sAnk1 that can partially or completely compensate for the changes in binding caused by charge-switching mutations in obscurin. We used molecular modeling to develop structural models of residues 6322-6339 of obscurin bound to sAnk1. The models, based on a combination of Brownian and molecular dynamics simulations, predict that the binding site on sAnk1 for obscurin is organized as two ankyrin-like repeats, with the last α-helical segment oriented at an angle to nearby helices, allowing lysine 6338 of obscurin to form an ionic interaction with aspartate 111 of sAnk1. This prediction was validated by double-mutant cycle experiments. Our results are consistent with a model in which electrostatic interactions between specific pairs of side chains on obscurin and sAnk1 promote binding and complex formation.     

Mapping the binding site on small ankyrin 1 for obscurin

Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.     

Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover

Protein turnover through cullin-3 is tightly regulated by posttranslational modifications, the COP9 signalosome, and BTB/POZ-domain proteins that link cullin-3 to specific substrates for ubiquitylation. In this paper, we report how potassium channel tetramerization domain containing 6 (KCTD6) represents a novel substrate adaptor for cullin-3, effectively regulating protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5). Binding of sAnk1.5 to KCTD6, and its subsequent turnover is regulated through posttranslational modification by nedd8, ubiquitin, and acetylation of C-terminal lysine residues. The presence of the sAnk1.5 binding partner obscurin, and mutation of lysine residues increased sAnk1.5 protein levels, as did knockdown of KCTD6 in cardiomyocytes. Obscurin knockout muscle displayed reduced sAnk1.5 levels and mislocalization of the sAnk1.5/KCTD6 complex. Scaffolding functions of obscurin may therefore prevent activation of the cullin-mediated protein degradation machinery and ubiquitylation of sAnk1.5 through sequestration of sAnk1.5/KCTD6 at the sarcomeric M-band, away from the Z-disk-associated cullin-3. The interaction of KCTD6 with ankyrin-1 may have implications beyond muscle for hereditary spherocytosis, as KCTD6 is also present in erythrocytes, and erythrocyte ankyrin isoforms contain its mapped minimal binding site.     

Obscurin is a ligand for small ankyrin 1 in skeletal muscle

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.     

Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle

Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ∼2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca²⁺ affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca²⁺ in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca²⁺ misregulation.     

Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC₅₀: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (K_D: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5.     

Contributions of the LPPVK motif of the iron-sulfur template protein IscU to interactions with the Hsc66-Hsc20 chaperone system

Hsc66 (HscA) and Hsc20 (HscB) from Escherichia coli comprise a specialized chaperone system that selectively binds the iron-sulfur cluster template protein IscU. Hsc66 interacts with peptides corresponding to a discrete region of IscU including residues 99-103 (LPPVK), and a peptide containing residues 98-106 stimulates Hsc66 ATPase activity in a manner similar to IscU. To determine the relative contributions of individual residues in the LPPVK motif to Hsc66 binding and regulation, we have carried out an alanine mutagenesis scan of this motif in the Glu98-Cys106 peptide and the IscU protein. Alanine substitutions in the Glu98-Cys106 peptide resulted in decreased ATPase stimulation (2-10-fold) because of reduced binding affinity, with peptide(P101A) eliciting <10% of the parent peptide stimulation. Alanine substitutions in the IscU protein also revealed lower activities resulting from decreased apparent binding affinity, with the greatest changes in Km observed for the Pro101 (77-fold), Val102 (4-fold), and Lys103 (15-fold) mutants. Calorimetric studies of the binding of IscU mutants to the Hsc66.ADP complex showed that the P101A and K103A mutants also exhibit decreased binding affinity for the ADP-bound state. When ATPase stimulatory activity was assayed in the presence of the co-chaperone Hsc20, each of the mutants displayed enhanced binding affinity, but the P101A and V102A mutants exhibited decreased ability to maximally simulate Hsc66 ATPase. A charge mutant containing the motif sequence of NifU, IscU(V102E), did not bind the ATP or ADP states of Hsc66 but did bind Hsc20 and weakly stimulated Hsc66 ATPase in the presence of the co-chaperone. These results indicate that residues in the LPPVK motif are important for IscU interactions with Hsc66 but not for the ability of Hsc20 to target IscU to Hsc66. The results are discussed in the context of a structural model based on the crystallographic structure of the DnaK peptide-binding domain.     

Requirement of membrane-proximal amino acids in the carboxyl-terminal tail for expression of the rat AT1a angiotensin receptor

A series of deletion mutants was created to analyze the function of the membrane-proximal region of the cytoplasmic tail of the rat type 1a (AT_1a) angiotensin receptor. In transiently transfected COS-7 cells, the truncated mutant receptors showed a progressive decrease in surface expression, with no major change in binding affinity for the peptide antagonist, [Sar¹,Ile⁸]angiotensin II. In parallel with the decrease in receptor expression, a progressive decrease in angiotensin II-induced inositol phosphate responses was observed. Alanine substitutions in the region 307–311 identified the highly conserved phenylalanine³⁰⁹ and adjacent lysine residues as significant determinants of AT_1a receptor expression.     

Some insights into the binding mechanism of the GABAA receptor: a combined docking and MM-GBSA study

Gamma-aminobutyric type A receptor (GABA_AR) is a member of the Cys-loop family of pentameric ligand gated ion channels (pLGICs). It has been identified as a key target for many clinical drugs. In the present study, we construct the structure of human 2α₁2β₂γ₂ GABA_AR using a homology modeling method. The structures of ten benzodiazepine type drugs and two non-benzodiazepine type drugs were then docked into the potential benzodiazepine binding site on the GABA_AR. By analyzing the docking results, the critical residues His102 (α₁), Phe77 (γ₂) and Phe100 (α₁) were identified in the binding site. To gain insight into the binding affinity, molecular dynamics (MD) simulations were performed for all the receptor–ligand complexes. We also examined single mutant GABA_AR (His102A) in complexes with the three drugs (flurazepam, eszopiclone and zolpidem) to elucidate receptor–ligand interactions. For each receptor–ligand complex (with flurazepam, eszopiclone and zolpidem), we calculated the average distance between the C_α of the mutant residue His102A (α₁) to the center of mass of the ligands. The results reveal that the distance between the C_α of the mutant residue His102A (α₁) to the center of flurazepam is larger than that between His102 (α₁) to flurazepam in the WT type complex. Molecular mechanic-generalized Born surface area (MM-GBSA)-based binding free energy calculations were performed. The binding free energy was decomposed into ligand-residue pairs to create a ligand-residue interaction spectrum. The predicted binding free energies correlated well (R ²?=?0.87) with the experimental binding free energies. Overall, the major interaction comes from a few groups around His102 (α₁), Phe77 (γ₂) and Phe100 (α₁). These groups of interaction consist of at least of 12 residues in total with a binding energy of more than 1 kcal mol^?1. The simulation study disclosed herein provides a meaningful insight into GABA_AR–ligand interactions and helps to arrive at a binding mode hypothesis with implications for drug design.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Molecular characterization of a thermostable L-fucose isomerase from Dictyoglomus turgidum that isomerizes L-fucose and D-arabinose

A recombinant thermostable l-fucose isomerase from Dictyoglomus turgidum was purified with a specific activity of 93 U/mg by heat treatment and His-trap affinity chromatography. The native enzyme existed as a 410 kDa hexamer. The maximum activity for l-fucose isomerization was observed at pH 7.0 and 80 °C with a half-life of 5 h in the presence of 1 mM Mn²⁺ that was present one molecular per monomer. The isomerization activity of the enzyme with aldose substrates was highest for l-fucose (with a k_cat of 15,500 min⁻¹ and a K_m of 72 mM), followed by d-arabinose, d-altrose, and l-galactose. The 15 putative active-site residues within 5 Å of the substrate l-fucose in the homology model were individually replaced with other amino acids. The analysis of metal-binding capacities of these alanine-substituted variants revealed that Glu349, Asp373, and His539 were metal-binding residues, and His539 was the most influential residue for metal binding. The activities of all variants at 349 and 373 positions except for a dramatically decreased k_cat of D373A were completely abolished, suggesting that Glu349 and Asp373 were catalytic residues. Alanine substitutions at Val131, Met197, Ile199, Gln314, Ser405, Tyr451, and Asn538 resulted in substantial increases in K_m, suggesting that these amino acids are substrate-binding residues. Alanine substitutions at Arg30, Trp102, Asn404, Phe452, and Trp510 resulted in decreases in k_cat, but had little effect on K_m.     

Molecular mechanism of the selectivity between BAFF/APRIL and their receptors by molecular simulations

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) belonging to the tumour necrosis factor (TNF) ligand family can bind three unusual TNF receptors (BCMA, TACI and BR3) with various binding affinities. BAFF and APRIL are regarded as promising therapeutic targets for autoimmune diseases because of their pivotal roles in cell survival and immune regulation. In this work, we carried out molecular dynamics calculations to explore the structural and chemical features responsible for ligand recognition by extracellular functional segments of TNF receptors. We found that the conserved pocket D_cons of BAFF/APRIL contacted the DxL motif of TNF receptors, while the D_spe1–3 sub-domains were responsible for their different affinities, especially D_spe1 and D_spe2. The residues at position II–V of DxL motif were wrapped into the D_cons pocket via salt-bridge and hydrophobic interactions. The hydrophobic residues of strand3 and helix1 in TNF receptors provided remarkable contributions for the affinities to BAFF/APRIL. Additionally, Arg^VI of DxL motif played a key role in the binding selectivity via salt-bridge interaction with residue D275B in BAFF. Arg27 in BCMA contributed to the high affinity for APRIL so that BCMA showed a preference for APRIL. Our studies indicated that Arg84 and Gln95 in TACI2 played an important role in the selectivity of two cysteine-rich domain segments in TACI, leading to the higher binding affinities of TACI2 than those of TACI1. The primary cause of the disability to bind APRIL was the space conflict with the rigid conformation of the C-terminus coil of BR3. These thorough understanding of the molecular mechanism for BAFF/APRIL recognition by their receptors provides new insights for guiding inhibitor design.     

Crystal structure of the MrkD1P receptor binding domain of Klebsiella pneumoniae and identification of the human collagen V binding interface

Klebsiella species are members of the family enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. Among other virulence factors in Klebsiella, type 3 pili exhibit a unique binding pattern in the human kidney via interaction of two MrkD adhesion variants 1C1 and 1P to type IV and/or V collagen. However, very little is known about the nature of this recognition. Here we present the crystal structure of the plasmid born MrkD_1P receptor domain (MrkDrd). The structure reveals a jelly‐roll β‐barrel fold comprising 17 β‐strands very similar to the receptor domain of GafD, the tip adhesin from the F17 pilus that recognizes n ‐acetyl‐d ‐glucosamine (GlcNAc). Analysis of collagen V binding of different MrkD_1P mutants revealed that two regions were responsible for its binding: a pocket, that aligns approximately with the GlcNAc binding pocket of GafD involving residues R105 and Y155, and a transversally oriented patch that spans strands β2a, β9b and β6 including residues V49, T52, V91, R102 and I136. Taken together, these data provide structural and functional insights on MrkD_1P recognition of host cells, providing a tool for future development of rationally designed drugs with the prospect of blocking Klebsiella adhesion to collagen V.     

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein a and rabbit IgG

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A fromStaphylococcus aureus (termed SpA¹) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA¹ and IgG-Fc were examined by the individual substitution of the residues in SpA¹ involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA¹ proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbant assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG₁-Fc (Fc fragment of human IgG₁) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.     

Human IgA-binding Peptides Selected from Random Peptide Libraries: AFFINITY MATURATION AND APPLICATION IN IGA PURIFICATION*

Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1–A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K_d = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K_d = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.     

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing.     

The importance of valine 114 in ligand binding in β2‐adrenergic receptor

G‐protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. β₂‐Adrenergic receptor (β₂‐AR) is a well‐studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand‐binding region of β₂‐AR using the recently solved high‐resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure‐function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster β₂‐AR by site‐directed mutagenesis. We replaced V114 in hamster β₂‐AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G‐protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side‐chains but also the aryl‐ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in β₂‐AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.     

Cholinergic Binding to the Receptor Proteolipid from Torpedo Electroplax Separated by Ion Exchange Chromatography

Abstract

Proteolipids (i.e., hydrophobic proteins) have been extracted with chloroform-methanol (2:1) from lyophilized Torpedo electroplax, and fractionated on a DEAE-cellulose column. The elution system consisted of the same solvent and a gradient of the hydrophobic ion ptoluene sulfonate (0.1–100mM). The three fractions obtained (I, II and III) have different content of protein, lipid P, and reducing sugars. The amino acid composition shows a higher proportion of hydrophobic residues in I and more charged ones in fractions II and III. Polyacryl amide gel electrophoresis of fraction I shows a single major band of 39 kdaltons; in fractions II and III a major band of 42 kdaltons and fainter bands in the range 62–68 kdaltons are observed.

Fraction I has the highest specific binding for [³H]-acetylcholine (7.1 nmol/mg protein) and [³H]α-bungarotoxin (5.2 nmol/mg protein). The nicotinic nature of this proteolipid was demonstrated by blocking experiments. The Scatchard plot showed two affinity sites for [³H]-acetylcholine (Kd₁ = 3nM and Kd₂ = 1.1 μm). 4-(N-maleimido) pheny1 [³H]trimethylammonium specifically labeled the 39 kdaltons band.

The possible factors involved in the fractionation of proteolipids are discussed. The findings suggest that the 39 kdaltons polypeptide contains the receptor site for acetylcholine and that the other proteolipid components may play a different function in the membrane.