Wheel Projection Research Articles

Cloning, expression, isotope labeling, purification, and characterization of bovine antimicrobial peptide, lactophoricin in Escherichia coli

Lactophoricin (LPcin-I) is a 23-amino acid peptide that corresponds to the carboxyterminal 113–135 region of component-3 of proteose peptone (PP3), a minor phosphoglycoprotein found in bovine milk. It has been reported that lactophoricin has antibacterial activity and a cationic amphipathic helical structure, but its shorter analogous peptide (LPcin-II), a 17-amino acid peptide, corresponding to the 119–135 region of PP3 does not display antibacterial activity. LPcin-I and LPcin-II have similar charge ratios and identical hydrophobic/hydrophilic sectors, according to their helical wheel projection patterns, and both peptides show cationic amphipathic helical folding and interact with membranes. However, it is known that only LPcin-I incorporates into planar lipidic bilayers to form voltage-dependent channels. In this study, the authors cloned and expressed the two recombinant peptides as ketosteroid isomerase (KSI) fusion proteins inclusion bodies in Escherichia coli. These peptides were subjected to NMR structural studies to explore their structure-activity relationships. Fusion proteins were purified by Ni–NTA affinity chromatography under denaturing conditions, and recombinant LPcin-I and LPcin-II were released from fusion by CNBr cleavage. Final purifications of LPcin-I and LPcin-II were achieved by preparative reversed-phase high performance liquid chromatography. Using these methods, we obtained several tens of milligrams of uniformly and selectively 15N labeled peptides per liter of growth, which was sufficient for solid-state NMR spectroscopy. Peptides were identified by tris-tricine polyacrylamide gel electrophoresis and HSQC spectra. Initial structural data were obtained by solution NMR spectroscopy and compared in membrane-like environments.

Domain Mapping of the Polycystin-2 C-terminal Tail Using de Novo Molecular Modeling and Biophysical Analysis

In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca2+) by isothermal titration calorimetry and undergo Ca2+-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca2+ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca2+-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease.

Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.

Analysis of secondary structures of odorant receptors

The structure‐function relationship of rhodopsin family GPCRs is well studied whereas little is known about the family of G protein‐coupled odorant receptors (ORs). Here the amino acid sequences and the functional domains of 385 human ORs were analyzed using AlignX, transmembrane (TM) helix prediction, and helix wheel projection. Most importantly, the critical residues, motifs and domains of ORs were examined by comparison with the corresponding ones of the rhodopsin family, especially the bioamine subfamily GPCRs that bind ligands with small size. Our result shows that as many as 59 residues in the TM domains and 33 residues in the non‐TM domains are conserved in >90% of ORs, an increase of 36.1% over 36 residues in the TM domains of the rhodopsin family GPCRs. Interestingly, the distributions of the highly conserved residues are largely distinct between the two family GPCRs, especially, in TM2, TM3, TM4, TM6 and TM7. The TM3 of ORs bearing highly conserved DRY motif for activation of Gα proteins was found to be one residue longer than that of the rhodopsin family GPCRs. These results suggest that ORs may have unique, but rather conserved global conformation and utilize structural mechanisms distinct from GPCRs of the rhodopsin family for ligand binding and activation.Supported by ASPET SURF award to MN and NIH grant (HL065492) to YHF

Residues 334 and 338 in Transmembrane Segment 8 of Human Equilibrative Nucleoside Transporter 1 Are Important Determinants of Inhibitor Sensitivity, Protein Folding, and Catalytic Turnover

Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.

Structure and Function Analysis of CaMdr1p, a Major Facilitator Superfamily Antifungal Efflux Transporter Protein of Candida albicans : Identification of Amino Acid Residues Critical for Drug/H + Transport

We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.

Aggregation of the Amphipathic Peptides (AAKA)ninto Antiparallel β-Sheets

Helical wheel projections of peptides based on the repeating unit Ac-(AAKA)n-NH2 clearly illustrate an amphipathic nature. One should therefore expect these peptides to form helices if the number of residues exceeds a certain threshold value. Indeed, ECD measurements show that Ac-(AAKA)4-NH2 and, to a minor extent, also Ac-(AAKA)3-NH2 exhibit some helical content at millimolar concentrations in aqueous solution. Surprisingly, however, these peptides were found to form hydrogels with an antiparallel beta-sheet conformation at centimolar concentrations. This occurs despite the positively charged lysine side chain which would be expected to inhibit the formation of extended beta-sheet layers.

The Plant Plasma Membrane Ca2+ Pump ACA8 Contains Overlapping as Well as Physically Separated Autoinhibitory and Calmodulin-binding Domains

In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.

Substituted Cysteine Accessibility of the Third Transmembrane Domain of the Creatine Transporter: DEFINING A TRANSPORT PATHWAY

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining approximately 75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na+ and Cl- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an alpha-helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321-28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na+- and Cl- -dependent neurotransmitter transporters.

Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2).

Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an approximately 200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1-832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RI alpha. Coimmunoprecipitation experiments confirmed interaction of in vitro translated BIG2 and RI alpha, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27-48 (domain A) interacted with RI alpha and RI beta, 284-301 (domain B) interacted with RII alpha and RII beta, and 517-538 (domain C) interacted with RI alpha, RII alpha, and RII beta. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.

Transmembrane domains in the functions of Fc receptors

In the present study, we use a novel method, PHDhtm, to predict the exact locations and extents of the transmembrane (TM) domains of multisubunit immunoglobulin Fc-receptors. Whereas most previous studies have used single residue hydrophobicity plots for characterizing of these domains, PHDhtm utilizes a system of neural networks and the evolutionary information contained in multiple alignments of related sequences to predict the above. Present PHDhtm application predicts TM domains of immunoglobulin Fc-receptors that in many cases differ significantly from those derived by using earlier methods. Comparisons of helical wheel projections of the presently derived TM domains from PHDhtm with those produced earlier reveal different hydrophobic moments as well as hydrophobic and hydrophilic surfaces. These differences probably alter the character of subunit association within the receptor complexes. This new algorithm can also be used for other membrane protein complexes and may advance both understanding the principles underlying such complexes formation and design of peptides that can interfere with such TM domain association so as to modulate specific cellular responses.

A Simple Approach to Membrane Protein Secondary Structure and Topology based on NMR Spectroscopy

This paper describes a simple, qualitative approach for the determination of membrane protein secondary structure and topology in lipid bilayer membranes. The approach is based on the observation of wheel-like resonance patterns observed in the NMR 1H- 15N/ 15N polarization inversion with spin exchange at the magic angle (PISEMA) and 1H/ 15N heteronuclear correlation (HETCOR) spectra of membrane proteins in oriented lipid bilayers. These patterns, named Pisa wheels, have been previously shown to reflect helical wheel projections of residues that are characteristic of α-helices associated with membranes. This study extends the analysis of these patterns to β-strands associated with membranes and demonstrates that, as for the case of α-helices, Pisa wheels are extremely sensitive to the tilt, rotation, and twist of β-strands in the membrane. Therefore, the Pisa wheels provide a sensitive, visually accessible, qualitative index of membrane protein secondary structure and topology.

Primary and secondary structures of grammistins, peptide toxins isolated from the skin secretion of the soapfish Pogonoperca punctata

SUMMARY: Six peptides (grammistins Pp 1, 2a, 2b, 3, 4a and 4b) with both hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Pogonoperca punctata by gel filtration and reverse-phase high performance liquid chromatography. They were completely sequenced and found to be simple peptides differing from the previous suggestion that they are peculiar peptides with tertiary amine or quaternary ammonium base moieties. Grammistins Pp 1, 2a and 2b were composed of 13 residues, Pp 3 of 25 residues and Pp 4a and 4b of 24 residues. While grammistins Pp 4a and 4b were identical and highly homologous with grammistin Gs 2 previously isolated from another soapfish Grammistes sexlineatus, the other grammistins did not have homologies with any proteinic or peptidic toxins known to date. Judging from circular dichroism data, helical wheel projections and hydrophobic moment plots, all the isolated grammistins are surface-seeking peptides with an abundance in amphiphilic α-helicity, similar to pardaxins from the skin secretion of two species of soles and melittin from bee venom as well as the G. sexlineatus grammistins.

An Early Ethylene Up-regulated Gene Encoding a Calmodulin-binding Protein Involved in Plant Senescence and Death

35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

A solid-state NMR index of helical membrane protein structure and topology.

The secondary structure and topology of membrane proteins can be described by inspection of two-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift polarization inversion spin exchange at the magic angle spectra obtained from uniformly (15)N-labeled samples in oriented bilayers. The characteristic wheel-like patterns of resonances observed in these spectra reflect helical wheel projections of residues in both transmembrane and in-plane helices and hence provide direct indices of the secondary structure and topology of membrane proteins in phospholipid bilayers. We refer to these patterns as PISA (polarity index slant angle) wheels. The transmembrane helix of the M2 peptide corresponding to the pore-lining segment of the acetylcholine receptor and the membrane surface helix of the antibiotic peptide magainin are used as examples.

Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein.

The E1 protein of bovine papillomavirus (BPV) is a site-specific DNA binding protein that recognizes an 18-bp inverted repeat element in the viral origin of replication. Sequence-specific DNA binding function maps to the region from approximately amino acids 140 to 300, and isolated polypeptides containing this region have been shown to retain origin binding in vitro. To investigate the sequence and structural characteristics which contribute to sequence-specific binding, the primary sequence of this region was examined for conserved features. The BPV E1 DNA binding domain (E1DBD) contains three major hydrophilic domains (HR1, amino acids 179-191; HR2, amino acids 218 to 230; and HR3, amino acids 241 to 252), of which only HR1 and HR3 are conserved among papillomavirus E1 proteins. E1DBD proteins with lysine-to-alanine mutations in HR1 and HR3 were severely impaired for DNA binding function in vitro, while a lysine-to-alanine mutation in HR2 had a minimal effect on DNA binding. Mutation of adjacent threonine residues in HR1 (T187 and T188) revealed that these two amino acids made drastically different contributions to DNA binding, with the T187 mutant being severely defective for origin binding whereas the T188 mutant was only mildly affected. Helical wheel projections of HR1 predict that T187 is on the same helical face as the critical lysine residues whereas T188 is on the opposing face, which is consistent with their respective contributions to DNA binding activity. To examine E1 binding in vivo, a yeast one-hybrid system was developed. Both full-length E1 and the E1DBD polypeptide were capable of specifically interacting with the E1 binding site in the context of the yeast genome, and HR1 was also critical for this in vivo interaction. Overall, our results indicate that HR1 is essential for origin binding by E1, and the features and properties of HR1 suggest that it may be part of a recognition sequence that mediates specific E1-nucleotide contacts.

Isolation and structures of grammistins, peptide toxins from the skin secretion of the soapfish Grammistes sexlineatus

Two peptide toxins (named grammistins Gs 1 and Gs 2) with hemolytic and ichthyotoxic activities were isolated from the skin secretion of the soapfish Grammistes sexlineatus. Grammistin Gs 2 showed 6–11× higher hemolytic activity and 10× higher ichthyotoxicity than grammistin Gs 1. The complete amino acid sequences of Gs 1 comprising 25 residues and Gs 2 comprising 24 residues were determined. Although a search by the database failed to find any homologous toxins from other sources, the grammistins were similar in secondary structures as well as biological activities to the two classes of peptide toxins, melittin from the bee venom and pardaxins from the skin secretion of two species of soles. CD experiments and helical wheel projections showed that the grammistins were randomly coiled in distilled water but formed amphiphilic α-helices in the presence of SDS micelles. In addition, they were found to be surface seeking peptides by the Eisenberg plot and assumed to exist as aggregates of 3–4 molecules. Interestingly, grammistin Gs 2 is much more abundant in amphiphilic α-helices and much higher in biological activities than melittin and pardaxins as well as grammistin Gs 1.

Helical structure and packing orientation of the S2 segment in the Shaker K+ channel.

Six transmembrane segments, S1-S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an alpha-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane alpha helix and that the high-impact face packs against other transmembrane segments in the functional channel.

Identification of Tethering Domains for Protein Kinase A Type Iα Regulatory Subunits on Sperm Fibrous Sheath Protein FSC1

The fibrous sheath is a unique cytoskeletal structure in the sperm flagellum believed to modulate sperm motility. FSC1 is the major structural protein of the fibrous sheath. The yeast two-hybrid system was used to identify other proteins that contribute to the structure of the fibrous sheath or participate in sperm motility. When FSC1 was used as the bait to screen a mouse testis cDNA library, two clones were isolated encoding the type Ialpha regulatory subunit (RIalpha) of cAMP-dependent protein kinase. Deletion analysis using the yeast two-hybrid system and in vitro binding assays with glutathione S-transferase-FSC1 fusion proteins identified two RIalpha tethering domains on FSC1. A domain located at residues 219-232 (termed domain A) corresponds to the reported tethering domain for a type II regulatory subunit (RII) of cAMP-dependent protein kinase, indicating that this binding domain has dual specificity to RI and RII. Another RIalpha tethering site (termed domain B) at residues 335-344 shows specific binding of RIalpha and had no significant sequence homology with known RII tethering domains. However, helical wheel projection analysis indicates that domain B is likely to form an amphipathic helix, the secondary structure of RII tethering domains of protein kinase A anchoring proteins. This was supported by the finding that site-directed mutagenesis to disrupt the amphipathic helix eliminated RIalpha binding. This is apparently the first report of an RIalpha-specific protein kinase A anchoring protein tethering domain.

Molecular Mechanics Modeling of the Interactions between the Active Domain of CAP18106–137 and Lipid A

AbstractIn this work, a computer search with molecular mechanics calculations is applied to determine the possible low‐energy configurations of two adjacent molecules, CAP18106–137 and lipid A. This computer search is performed by systematically searching the five degrees of freedom that define the relative orientations of the two adjacent molecules. Hydrophilic and hydrophobic units are separated into several small domains along the CAP18106–137 molecule so that it can interact with the lipid A either through the Coulombic interactions with the diphosphoryl head group or through the hydrophobic interactions with the fatty acyl chains. The intermolecular interactions are calculated through the van der Waals and Coulombic interactions. The Coulombic interactions are calculated by using a dielectric constant either with a value of unity ϵ = 1 or with a distance‐dependent dielectric function ϵ(r) = rij. Based on the 400 lowest searched configurations, the intermolecular interactions calculated with ϵ = 1 are found to be about sevenfold lower than those calculated with ϵ(r) = rij. Based on the inspection of the sixteen snapshots of molecular associations, the hydrophobic interactions calculated with ϵ(r) = rij are more favorable than those calculated with ϵ = 1. The high population of the lipid A binding to the CAP 18106–137 molecule is located within the first 19 residues near the N‐terminus of the peptide, as calculated either with e = 1 or with ϵ(r) = rij, by observing the diphosphoryl groups of the lipid A. The orientations of the lipid A with respect to the CAP18106–137 molecule complete an angle of 260°, which appose to the positively charged residues in the N‐terminal half by using the helical wheel projection of the peptide.