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1.
Biochemistry ; 56(41): 5503-5511, 2017 10 17.
Article in English | MEDLINE | ID: mdl-28930470

ABSTRACT

Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.


Subject(s)
HIV Envelope Protein gp41/metabolism , Models, Molecular , Amino Acid Sequence , Conserved Sequence , Crystallography, X-Ray , Databases, Protein , Epitopes , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/genetics , Hydrophobic and Hydrophilic Interactions , Micelles , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistry , Phosphorylcholine/metabolism , Protein Conformation , Protein Conformation, alpha-Helical , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solid-Phase Synthesis Techniques , Tandem Repeat Sequences
2.
J Biol Chem ; 292(9): 3591-3602, 2017 03 03.
Article in English | MEDLINE | ID: mdl-28100777

ABSTRACT

During sexual development ascomycete fungi produce two types of peptide pheromones termed a and α. The α pheromone from the budding yeast Saccharomyces cerevisiae, a 13-residue peptide that elicits cell cycle arrest and chemotropic growth, has served as paradigm for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available for α pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S. cerevisiae homolog. High resolution structure of synthetic α-pheromone from the plant pathogenic ascomycete Fusarium oxysporum revealed the presence of a central ß-turn resembling that of its yeast counterpart. Disruption of the-fold by d-alanine substitution of the conserved central Gly6-Gln7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in chemoattractant activity. Unexpectedly, the growth inhibitory effect of F. oxysporum α-pheromone was independent of the cognate G protein-coupled receptors Ste2 and of the central ß-turn but instead required two conserved Trp1-Cys2 residues at the N terminus. These results indicate that, despite their reduced size, fungal α-pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as polarity reorientation and cell division.


Subject(s)
Chemotactic Factors/chemistry , Fungal Proteins/chemistry , Fusarium/chemistry , Pheromones/chemistry , Cell Cycle , Cell Nucleus/metabolism , Cysteine/chemistry , Genes, Mating Type, Fungal , Peptides/chemistry , Protein Domains , Protein Structure, Secondary , Receptors, G-Protein-Coupled/metabolism , Saccharomyces cerevisiae/chemistry , Signal Transduction , Structure-Activity Relationship , Tryptophan/chemistry
3.
J Biol Chem ; 290(21): 12999-3015, 2015 May 22.
Article in English | MEDLINE | ID: mdl-25787074

ABSTRACT

The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.


Subject(s)
HIV Antibodies/immunology , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/immunology , HIV Infections/immunology , Membrane Fusion/immunology , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Circular Dichroism , Crystallography, X-Ray , HIV Envelope Protein gp41/metabolism , HIV Infections/virology , HIV-1/physiology , Humans , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Molecular Sequence Data , Peptide Fragments/metabolism , Protein Structure, Secondary , Rabbits , Virus Internalization
4.
J Biol Chem ; 289(10): 6565-6580, 2014 Mar 07.
Article in English | MEDLINE | ID: mdl-24429284

ABSTRACT

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.


Subject(s)
AIDS Vaccines/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , HIV Envelope Protein gp41/immunology , Immunodominant Epitopes/immunology , AIDS Vaccines/chemistry , Amino Acid Sequence , Broadly Neutralizing Antibodies , HIV Antibodies , HIV Envelope Protein gp41/chemistry , Humans , Immunodominant Epitopes/chemistry , Micelles , Molecular Sequence Data , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistry , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrophotometry, Infrared , Vaccines, Subunit/chemistry , Vaccines, Subunit/metabolism
5.
PLoS One ; 8(1): e54568, 2013.
Article in English | MEDLINE | ID: mdl-23349931

ABSTRACT

Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease's activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.


Subject(s)
HIV Protease/chemistry , Host-Parasite Interactions , Protein Conformation , Ribonucleases/chemistry , Amino Acid Sequence , Enzyme Stability , HIV Infections/enzymology , HIV Infections/pathology , HIV Protease/genetics , HIV-1/chemistry , Humans , Infections/enzymology , Infections/pathology , Magnetic Resonance Spectroscopy , Protein Folding , Protein Structure, Secondary , Ribonucleases/genetics
6.
J Biomol NMR ; 55(3): 231-7, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23314728

ABSTRACT

Intrinsically disordered proteins (IDPs) have recently attracted the attention of the scientific community challenging the well accepted structure-function paradigm. In the characterization of the dynamic features of proteins nuclear magnetic resonance spectroscopy (NMR) is a strategic tool of investigation. However the peculiar properties of IDPs, with the lack of a unique 3D structure and their high flexibility, have a strong impact on NMR observables (low chemical shift dispersion, efficient solvent exchange broadening) and thus on the quality of NMR spectra. Key aspects to be considered in the design of new NMR experiments optimized for the study of IDPs are discussed. A new experiment, based on direct detection of (13)C(α), is proposed.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Proteins/chemistry , Humans , Protein Conformation
7.
Biomol NMR Assign ; 7(1): 13-5, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22392335

ABSTRACT

Onconase(®) FL-G zymogen is a 120 residue protein produced by circular permutation of the native Onconase(®) sequence. In this construction, the wild type N- and C-termini are linked by a 16 residue segment and new N- and C-termini are generated at wild type positions R73 and S72. This novel segment linking the native N- and C-termini is designed to obstruct Onconase's(®) active site and encloses a cleavage site for the HIV-1 protease. As a first step towards the resolution of its 3D structure and the study of its structure-function relationships, we report here the nearly complete NMR (1)H, (13)C and (15)N resonance chemical shift assignments at pH 5.2 and 35°C (BMRB deposit no 17973). The results presented here clearly show that the structure of the wild type Onconase(®) is conserved in the FL-G zymogen.


Subject(s)
Enzyme Precursors/chemistry , Nuclear Magnetic Resonance, Biomolecular , Ribonucleases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Enzyme Precursors/metabolism , HIV Protease/metabolism , Ribonucleases/metabolism
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