Heptad Repeat Motif Research Articles

Association of a Model Transmembrane Peptide Containing Gly in a Heptad Sequence Motif

A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A.

Understanding Metalloprotein Folding Using a de Novo Design Strategy

Metal ions play significant roles in most biological systems. Over the past two decades, there has been significant interest in the redesign of existing metal binding sites in proteins/peptides and the introduction of metals into folded proteins/peptides. Recent research has focused on the effects of metal binding on the overall secondary and tertiary conformations of unstructured peptides/proteins. In this context, de novo design of metallopeptides has become a valuable approach for studying the consequence of metal binding. It has been seen that metal ions not only direct folding of partially folded peptides but have at times also been the elixir for properly folding random-coil-like structures in stable secondary conformations. Work in our group has focused on binding of heavy metal ions such as Hg(II) to de novo designed alpha-helical three stranded coiled coil peptides with sequences based on the heptad repeat motif. Removal from or addition of a heptad to the parent 30-residue TRI peptide with the amino acid sequence Ac-G(LKALEEK)(4)G-NH(2) generated peptides whose self-aggregation affinities were seen to be dependent on their lengths. It was noted that adjustment in the position of the thiol from an "a" position in the case of the shorter BabyL9C to a "d" position for BabyL12C resulted in a peptide with low association affinities for itself, weaker binding with Hg(II), and a considerably faster kinetic profile for metal insertion. Similar differences in thermodynamic and kinetic parameters were also noted for the longer TRI peptides. At the same time, metal insertion into the prefolded and longer TRI and Grand peptides has clearly demonstrated that the metal binding is both thermodynamically as well kinetically different from that to unassociated peptides.

Evaluation of the molecular interactions of fluorinated amino acids with native polypeptides.

One fluorine can make a difference. Investigation of the properties of fluorinated amino acids in a native-polypeptide environment increases our ability to use this class of non-natural amino acids for protein engineering. Two fluorinated amino acids (see figure) were incorporated into different positions of a heptad repeat motif, and their influence on coiled-coil peptide formation was studied. The screens introduced here are sufficiently sensitive to even detect the difference of one single fluorine atom in the side chain of an amino acid.

Direct Interaction of Major Histocompatibility Complex Class II-derived Peptides with Class Ia Phosphoinositide 3-Kinase Results in Dose-dependent Stimulatory Effects

Peptides corresponding to residues 65-79 of human lymphocyte antigen class II sequence (DQA*03011) are cell-permeable and at high concentrations block activation of protein kinase B/Akt and p70-S6 kinase in T-cells, effects attributed to inhibition of phosphoinositide (PI) 3-kinase activity. To understand the molecular basis of this, we analyzed the effect this peptide had on activity of class I PI 3-kinases. Although there was no effect on the activity of class Ib PI 3-kinase or on the protein kinase activity of class I PI 3-kinases, there was a biphasic effect on lipid kinase activity of the class Ia enzymes. There was an inhibition of activity at higher peptide concentrations because of a formation of insoluble complexes between peptide and enzyme. Conversely, at lower peptide concentrations there was a profound activation of PI 3-kinase activity of class Ia PI 3-kinases. Studies of peptide variants revealed that all active peptides conform to heptad repeat motifs characteristic of coiled-coil helices. Surface plasmon resonance studies confirmed direct sequence-specific binding of active peptide to the p85alpha adapter subunit of class Ia PI 3-kinase. Active peptides also activated protein kinase B and extracellular signal-regulated kinase (ERK) in vivo in a wortmannin-sensitive manner while reducing recoverable cellular p85 levels. These results indicate that the human lymphocyte antigen class II-derived peptides regulate PI 3-kinase by direct interaction, probably via the coiled-coil domain. These peptides define a novel mechanism of regulating PI 3-kinase and will provide a useful tool for specifically dissecting the function of class Ia PI 3-kinase in cells and for probing structure-function relationships in the class Ia PI 3-kinase heterodimers.

Six-helix bundle assembly and analysis of the central core of mumps virus fusion protein

The fusion protein of enveloped viruses mediates the fusion between the viral and cellular membranes, allowing the penetration of the viral genomes into the host cell. Many of these proteins share a common fold comprising a central core trimer of anti-parallel coiled-coil heterodimers, which are formed by two discontinuous heptad repeat (HR) motifs located at the ectodomain of the fusion proteins. In this study, we constructed and purified the corresponding regions (HR1 and HR2) of mumps virus fusion protein that are predicted to form coiled coil. The HR1 and HR2 were expressed and purified separately or as a single chain connected by an amino acid linker (HR1-linker-HR2, named 2-Helix). Series of biochemical and biophysical analyses of the expressed proteins have shown that HR1 and HR2 of mumps virus fusion protein share the common features of other enveloped virus fusion proteins. CD spectral results show that HR1 forms an α-helical coil structure while HR2 exists as an unstructured monomer in PBS in nature. Mixtures of HR1 and HR2 could form a stable six-helix bundle, indicating the interaction of HR1 and HR2. The 2-Helix protein also shows characteristic properties of the 6-helix bundle. Therefore, mumps virus fusion protein has a common core architecture and its HR regions could be used as a drug target for virus fusion inhibitors.

In Vitro Selection of Membrane-spanning Leucine Zipper Protein-Protein Interaction Motifs Using POSSYCCAT

A membrane-spanning heptad repeat motif mediates interaction between transmembrane segments. This motif was randomized with three different sets of mostly hydrophobic residues in the context of POSSYCCAT, a modified ToxR transcription activator system. The resulting combinatorial libraries were subjected to different levels of selective pressure to obtain groups of transmembrane segments that are distinguished by their ability to self-interact in bacterial membranes. Upon relating self-interaction to amino acid composition, the following conclusions were made. First, randomization with only Leu, Ile, Val, Met, and Phe resulted in unexpected robust self-interaction with little sequence specificity. Second, with more complex amino acid mixtures that represent natural transmembrane segments more closely, self-interaction critically depended on amino acid composition of the interface. Whereas the contents of Ile and Leu residues increased with the ability to self-interact, the contents of Pro and Arg residues decreased. Third, heptad repeat motifs composed of Leu, Ile, Val, Met, and Phe were approximately 40-fold over-represented in transmembrane segments of single-span membrane proteins as compared with motifs composed of the more complex amino acid mixtures. This suggests that heptad motifs composed of the smaller subset of amino acids were enriched in the course of natural single-span membrane protein evolution.

Peptides corresponding to the heptad repeat motifs in the transmembrane protein (gp41) of human immunodeficiency virus type 1 elicit antibodies to receptor-activated conformations of the envelope glycoprotein.

Two heptad repeat regions in the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane subunit (gp41) self-assemble into a six-helix bundle structure that is critical for virus entry. Immunizations with peptides corresponding to these regions generated antibodies specific to the receptor-activated conformations of gp41.

The Amino-terminal Heptad Repeats of the Coiled-coil Neck Domain of Pulmonary Surfactant Protein D Are Necessary for the Assembly of Trimeric Subunits and Dodecamers

Pulmonary surfactant protein D (SP-D), a lung host defense protein, is assembled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of trimers is required for many of its functions. A peptide containing the alpha-helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular assembly of disulfide-cross-linked, trimeric subunits and dodecamers. For the present studies, we synthesized mutant cDNAs with deletions or site-directed substitutions in the neck domain of rat SP-D, and examined the assembly of the newly synthesized proteins after transfection of CHO-K1 cells. The neck domain contains three "classical" heptad repeat motifs with leucine residues at the "d position," and a distinctive C-terminal repeat previously suggested to drive trimeric chain association. Deletion of the highly conserved core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. By contrast, deletion of the entire neck domain or deletion of one or two amino-terminal repeats resulted in defective molecular assembly. The secreted proteins eluted in the position of monomers by gel filtration under nondenaturing conditions. In addition, the neck + carbohydrate recognition domain of SP-D was necessary and sufficient for the trimerization of a heterologous collagen sequence located amino-terminal to the trimeric coiled-coil. These studies provide strong evidence that the amino-terminal heptad repeats of the neck domain are necessary for the intracellular, trimeric association of SP-D monomers and for the assembly and secretion of functional dodecamers.

The core of the respiratory syncytial virus fusion protein is a trimeric coiled coil.

Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (K(3) approximately 2.2 x 10(11) M(-2)) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with beta-sheet-like characteristics. Only a small increase in alpha-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309-319, 1999). Finally, HR3 forms weak alpha-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.

Mutational Analysis of β′260–309, a ς70 Binding Site Located on Escherichia coliCore RNA Polymerase

In eubacteria, the final sigma subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters. Previously, our laboratory defined a region of the beta' subunit that interacts with final sigma(70) in vitro. This region of beta' contained heptad repeat motifs indicative of coiled coils. In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260-309 of beta', to look at disruption of the final sigma(70)-core interaction. Several of the mutants were defective for binding final sigma(70) in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in which the mutant beta' is the sole source of beta' subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for Efinal sigma(70) holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor final sigma factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K. , and Darst, S. A. (1999) Cell 98, 811-824), the region homologous to beta'(260-309) of Escherichia coli forms a coiled coil. Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil.

A single amino acid change in the Newcastle disease virus fusion protein alters the requirement for HN protein in fusion.

The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F(1) portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.

Cell division in Escherichia coli: role of FtsL domains in septal localization, function, and oligomerization.

In Escherichia coli, nine essential cell division proteins are known to localize to the division septum. FtsL is a 13-kDa bitopic membrane protein with a short cytoplasmic N-terminal domain, a membrane-spanning segment, and a periplasmic domain that has a repeated heptad motif characteristic of leucine zippers. Here, we identify the requirements for FtsL septal localization and function. We used green fluorescent protein fusions to FtsL proteins where domains of FtsL had been exchanged with analogous domains from either its Haemophilus influenzae homologue or the unrelated MalF protein to show that both the membrane-spanning segment and the periplasmic domain of FtsL are required for localization to the division site. Mutagenesis of the periplasmic heptad repeat motif severely impaired both localization and function as well as the ability of FtsL to drive the formation of sodium dodecyl sulfate-resistant multimers in vitro. These results are consistent with the predicted propensity of the FtsL periplasmic domain to adopt a coiled-coiled structure. This coiled-coil motif is conserved in all gram-negative and gram-positive FtsL homologues identified so far. Our data suggest that most of the FtsL molecule is a helical coiled coil involved in FtsL multimerization.

A Single Amino Acid Change in the Newcastle Disease Virus Fusion Protein Alters the Requirement for HN Protein in Fusion

The role of a leucine heptad repeat motif between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or the fusion activity of the protein. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. L268A mutant protein was cleaved and expressed at the cell surface although the protein migrated slightly slower than wild type on polyacrylamide gels, suggesting an alteration in conformation or processing. L268A protein was fusion inactive in the presence or absence of HN protein expression. Mutant L289A protein was expressed at the cell surface and proteolytically cleaved at better than wild-type levels. Most importantly, this protein mediated syncytium formation in the absence of HN protein expression although HN protein enhanced fusion activity. These results show that a single amino acid change in the F1 portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation.

A discrete stage of baculovirus GP64-mediated membrane fusion.

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

Mutational analysis of heptad repeats in the membrane-proximal region of Newcastle disease virus HN protein.

For most paramyxoviruses, syncytium formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (X. Hu et al., J. Virol. 66:1528-1534, 1992). The stalk region of the Newcastle disease virus (NDV) HN protein has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (R. Buckland et al., J. Gen. Virol. 73:1703-1707, 1992; T. Sergel-Germano et al., J. Virol. 68:7654-7658, 1994; J. Reitter et al., J. Virol. 69:5995-6004, 1995). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic a-position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein activity and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.

Cleavage susceptibility of reovirus attachment protein sigma1 during proteolytic disassembly of virions is determined by a sequence polymorphism in the sigma1 neck.

A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein sigma1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes sigma1 cleavage upon conversion of virions to ISVPs. We examined the sigma1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and sigma1 cleavage. The sigma1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the sigma1 proteins of the remaining strains. Thr249 occupies the d position of a heptad repeat motif predicted to stabilize sigma1 oligomers through alpha-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of sigma1 known as the neck. Substitution of Thr249 with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D sigma1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the sigma1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves sigma1 after Arg245. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of sigma1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for sigma1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.

The MYND motif is required for repression of basal transcription from the multidrug resistance 1 promoter by the t(8;21) fusion protein.

Chromosomal translocations in acute leukemia that affect the AML-1/CBFbeta transcription factor complex create dominant inhibitory proteins. However, the mechanisms by which these proteins act remain obscure. Here we demonstrate that the multidrug resistance 1 (MDR-1) promoter is a target for AML/ETO transcriptional repression. This repression is of basal, not activated, expression from the MDR-1 promoter and thus represents a new mechanism for AML/ETO function. We have defined two domains in AML/ETO that are required for repression of basal transcription from the MDR-1 promoter: a hydrophobic heptad repeat (HHR) motif and a conserved zinc finger (ZnF) domain termed the MYND domain. The HHR mediates formation of AML/ETO homodimers and AML/ETO-ETO heterodimers. Single serine substitutions at conserved cysteine residues within the predicted ZnFs also abrogate transcriptional repression. Finally, we observe that AML/ETO can also inhibit Ets-1 activation of the MDR-1 promoter, indicating that AML/ETO can disrupt both basal and Ets-1-dependent transcription. The fortuitous inhibition of MDR-1 expression in t(8;21)-containing leukemias may contribute to the favorable response of these patients to chemotherapeutic drugs.

Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: potential role for coiled coil interaction.

Troponin T (TnT), a thin filament myofibrillar protein, is essential for the Ca2+ regulation of striated muscle contraction in vertebrates, both in vivo and in vitro. To understand the role of TnT in this process, its interaction with two other troponin components, troponin I (TnI) and troponin C (TnC) was examined by using the yeast two hybrid system, which is a genetic approach to detect protein-protein interactions. Computer assisted analysis of phylogenetically distant TnT amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential for alpha-helical coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TnI sequences. These protein motifs appeared to be the regions where TnI-TnT interaction may take place. Deletions and point mutations in TnT, which disrupted its HR motif, severely reduced or abolished TnI binding, but binding to TnC was not affected, indicating that the TnT-TnI and TnT-TnC binary interactions can be uncoupled. Remarkably, the truncated fragments of TnT and TnI in which the HR motifs were retained showed binary interaction in the yeast two hybrid system. It was also observed that the formation of the TnT-TnI heterodimers is favored over the homodimers TnT-TnT and TnI-TnI. These results indicate that the evolutionarily conserved HR motifs may play a role in TnT-TnI dimerization, presumably through the formation of alpha-helical coiled coils.

Determinants of human immunodeficiency virus type 1 resistance to gp41-derived inhibitory peptides.

A synthetic peptide, DP178, containing amino acids 127 to 162 of the human immunodeficiency virus type 1 (HIV-1) gp41 Env glycoprotein, is a potent inhibitor of virus infection and virus mediated cell-to-cell fusion (C. Wild, T. Greenwell, and T. Matthews, AIDS Res. Hum. Retroviruses 9:1051-1053, 1993). In an effort to understand the mechanism of action of this peptide, we derived resistant variants of HIV-1(IIIB) and NL4-3 by serial virus passage in the presence of increasing doses of the peptide. Sequence analysis of the resistant isolates suggested that a contiguous 3-amino-acid sequence within the amino-terminal heptad repeat motif of gp41 was associated with resistance. Site-directed mutagenesis studies confirmed this observation and indicated that changes in two of these three residues were necessary for development of the resistant phenotype. Direct binding of DP178 to recombinant protein and synthetic peptide analogs containing the wild-type and mutant heptad repeat sequences revealed a strong correlation between DP178 binding and the biological sensitivity of the corresponding virus isolates to DP178. The results are discussed from the standpoints of the mechanism of action of DP178 and recent crystallographic information for a core structure of the gp41 ectodomain.

Molecular Characterization of Mutants of the Acetate Regulatory GenefacBofAspergillus nidulans

Todd, R. B., Kelly, J. M., Davis, M. A., and Hynes, M. J. 1997. Molecular characterization of mutants of the acetate regulatory genefacBofAspergillus nidulans.22,92–102. The facB gene ofAspergillus nidulansencodes a DNA binding transcriptional activator required for growth on acetate as a sole carbon source. FacB contains N-terminal GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA binding and leucine zipper-like heptad repeat motifs and central and C-terminal acidic α-helical regions.facBrecessive loss of function mutants are deficient in acetate induction of acetyl-CoA synthase, isocitrate lyase, malate synthase, acetamidase, and NADP-isocitrate dehydrogenase. Characterization of lesions infacBmutant alleles has localized important functional regions of the FacB protein. Two extreme mutants are shown to lack the C-terminal region of the protein. Two temperature sensitive mutants contain amino acid substitutions in the DNA binding domain and are shown to affect acetate induction ofamdS–lacZexpression and confer temperature sensitivein vitroDNA binding. Two temperature sensitivefacBmutations result in thermolability of acetyl-CoA synthase, isocitrate lyase, and malate synthase but not acetamidase or NADP-isocitrate dehydrogenase in crude extracts. This suggests that FacB may have a structural role in acetate metabolism in addition to its regulatory function.