Structural investigation of the HIV-1 envelope glycoprotein gp160 cleavage site 3: role of site-specific mutations

Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date.In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure-activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2', while sharing the same N-terminal sequence, containing helix-breaking D-amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity.     

GLUE That Sticks to HIV: A Helix‐Grafted GLUE Protein That Selectively Binds the HIV gp41 N‐Terminal Helical Region

Methods for the stabilization of well‐defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Here, we report the stable and functional display of an HIV gp41 C‐peptide helix mimic on a G RAM‐L ike U biquitin‐binding in E AP45 (GLUE) protein. C‐peptide helix‐grafted GLUE selectively binds a mimic of the N‐terminal helical region of gp41, a well‐established HIV drug target, in a complex cellular environment. Additionally, the helix‐grafted GLUE is folded in solution, stable in human serum, and soluble in aqueous solutions, and thus overcomes challenges faced by a multitude of peptide drugs, including those derived from HIV gp41 C‐peptide.     

Exploring Viral Interference Using Peptides: Molecular Determinants of HIV-1 Inhibition by a Peptide Derived from Human Pegivirus-1 Envelope Protein E2

Co-infection with the human pegivirus 1 (HPgV-1) often has a beneficial effect on disease progression in HIV-1-infected individuals. Several HPgV-1 proteins and peptides, including a 20-mer peptide (P6-2) derived from the N-terminal region of the HPgV-1 surface protein E2, have been associated with this phenomenon, which is referred to as viral interference. We identified the cysteine residues, the hydrophobic core tetrapeptide, as well as the C-terminal negative charge as key factors for the HIV-1 inhibitory activity of P6-2. Analysis of mutations in P6-2-resistant HIV-1 indicated a binding site for the peptide in the HIV-1 envelope glycoprotein gp120. In fact, P6-2 was shown to bind to soluble gp120, as well as to a peptide presenting the gp120 V3 loop. Furthermore, the HIV-1 inhibitory activity of P6-2 could be revoked by the V3 loop peptide, thus indicating a molecular mechanism that involves interaction of P6-2 with the gp120 V3 loop.     

Peptide Fragments of Odin‐Sam1: Conformational Analysis and Interaction Studies with EphA2‐Sam

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin‐Sam1, binds to the Sam domain of the EphA2 receptor (EphA2‐Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin‐Sam1 associates with EphA2‐Sam by adopting a “mid‐loop/end‐helix” model. In this study three peptide sequences, encompassing the mid‐loop interacting portion of Odin‐Sam1 and its C‐terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2‐Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2‐Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti‐cancer therapeutics as inhibitors of EphA2‐Sam heterotypic interactions.     

Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors

We report the engineering of the monocyclic sunflower trypsin inhibitor (SFTI‐1[1,14]) into a potent furin inhibitor. In a rational approach, we converted the native scaffold of this trypsin‐like serine protease inhibitor into a subtilisin‐like one by substitutions in the canonical and, particularly, in the substrate‐binding loop. Although the substrate sequence for furin is Arg‐X‐Arg/Lys‐Arg↓, the most potent inhibitor had a lysine at position P1. C‐terminally truncated versions demonstrated the strongest activity, thus suggesting a lack of interaction between this motif and the surface of furin. This observation was further supported by molecular modeling. With an inhibition constant of 0.49 nm , the engineered peptide H‐KRCKKSIPPICF‐NH₂ is a promising compound for further development of furin inhibitors aimed at controlling the activity of this protease in vitro and in vivo.     

Chemoenzymatic synthesis of HIV-1 gp41 glycopeptides: effects of glycosylation on the anti-HIV activity and alpha-helix bundle-forming ability of peptide C34

C34 is a 34-mer peptide derived from the C-terminal ectodomain of HIV-1 envelope glycoprotein, gp41. The C34 region in native gp41 carries a conserved N-glycan at Asn637 and the sequence is directly involved in the virus-host membrane fusion, an essential step for HIV-1 infection. This paper describes the synthesis of glycoforms of C34 which carry a monosaccharide, a disaccharide, and a native oligosaccharide moiety. The synthesis of the glycopeptide which carries a native high-mannose type N-glycan was achieved by a chemoenzymatic approach by using an endoglycosidase-catalyzed oligosaccharide transfer as the key step. The effects of glycosylation on the inhibitory activity and the helix-bundle forming ability of C34 were investigated. It was found that glycosylation moderately decreases the anti-HIV activity of C34 and, in comparison with C34, glyco-C34 forms less compact six-helix bundles with the corresponding N-terminal peptide, N36. This study suggests that conserved glycosylation modulates the anti-HIV activity and conformations of the gp41 C-peptide, C34.     

Design of a Short Thermally Stable α‐Helix Embedded in a Macrocycle

Although helices play key roles in peptide–protein and protein–protein interactions, the helical conformation is generally unstable for short peptides (10–15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain‐to‐side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non‐hydrogen‐bonded amide groups to solvent. Here, we develop a “capped‐strapped” peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end‐capping interactions. We have designed a ten‐residue capped‐strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T_m≈70 °C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X‐ray crystallography revealed a novel quaternary structure with implications for foldamer design.     

Selective DNA‐Binding by Designed Bisbenzamidine‐Homeodomain Chimeras

We report the construction of conjugates between three variants of the helix 3 region of a Q50K engrailed homeodomain and bisbenzamidine minor‐groove DNA binders. The hybrid featuring the sequence of the native protein failed to bind to DNA; however, modifications that increased the α‐helical folding propensity of the peptide allowed specific DNA binding by a bipartite (major/minor groove) interaction.     

The LLSGIV stretch of the N-terminal region of HIV-1 gp41 is critical for binding to a model peptide, T20

A number of peptides and peptide analogs derived from the membraneproximal region of gp41 ectodomain are found to be effectiveinhibitors of human immunodeficiency virus type 1 (HIV-1)-mediatedfusion events. One of them, T20 (aa 638–673), was founddisordered and sparingly soluble in water, but became solubleupon mixing with selected, structured peptides from the aminoterminal heptad repeat (HR1) region of gp41 using a simple andsensitive method of reduction in the scattering of T20 suspension.From the results on mapping the locus of interaction with T20by employing partially overlapping peptides derived from HR1,it was concluded that the LLSGIV segment was a critical dockingsite for the C-terminal peptide of gp41 in its putative inhibitoryaction consistent with a previous fluorescence study. It wasalso found that peptides capable of solubilizing T20 dispersionhave a high content of helix, as well as ß-strand,conformation in aqueous solution. Specificity of T20/HR1-derivedpeptide binding was ascertained by using a scrambled sequenceof a T20-active peptide and a plateau in scattering reductionof T20 suspension with variation in the concentration of a T20-activeHR1 peptide. Implications on the mechanism of T20 inhibitionand the sequence of folding of the gp41 core structure are discussed.     

A hydrogen bond surrogate approach for stabilization of short peptide sequences in alpha-helical conformation

Alpha-helices constitute the largest class of protein secondary structures and play a major role in mediating protein-protein interactions. Development of stable mimics of short alpha-helices would be invaluable for inhibition of protein-protein interactions. This Account describes our efforts in developing a general approach for constraining short peptides in alpha-helical conformations by a main-chain hydrogen bond surrogate (HBS) strategy. The HBS alpha-helices feature a carbon-carbon bond derived from a ring-closing metathesis reaction in place of an N-terminal intramolecular hydrogen bond between the peptide i and i + 4 residues. Our approach is centered on the helix-coil transition theory in peptides, which suggests that the energetically demanding organization of three consecutive amino acids into the helical orientation inherently limits the stability of short alpha-helices. The HBS method affords preorganized alpha-turns to overcome this intrinsic nucleation barrier and initiate helix formation. The HBS approach is an attractive strategy for generation of ligands for protein receptors because placement of the cross-link on the inside of the helix does not block solvent-exposed molecular recognition surfaces of the molecule. Our metathesis-based synthetic strategy utilizes standard Fmoc solid phase peptide synthesis methodology, resins, and reagents and provides HBS helices in sufficient amounts for subsequent biophysical and biological analyses. Extensive conformational analysis of HBS alpha-helices with 2D NMR, circular dichroism spectroscopies and X-ray crystallography confirms the alpha-helical structure in these compounds. The crystal structure indicates that all i and i + 4 C=O and NH hydrogen-bonding partners fall within distances and angles expected for a fully hydrogen-bonded alpha-helix. The backbone conformation of HBS alpha-helix in the crystal structure superimposes with an rms difference of 0.75 A onto the backbone conformation of a model alpha-helix. Significantly, the backbone torsion angles for the HBS helix residues fall within the range expected for a canonical alpha-helix. Thermal and chemical denaturation studies suggest that the HBS approach provides exceptionally stable alpha-helices from a variety of short sequences, which retain their helical conformation in aqueous buffers at exceptionally high temperatures. The high degree of thermal stability observed for HBS helices is consistent with the theoretical predictions for a nucleated helix. The HBS approach was devised to afford internally constrained helices so that the molecular recognition surface of the helix and its protein binding properties are not compromised by the constraining moiety. Notably, our preliminary studies illustrate that HBS helices can target their expected protein receptors with high affinity.     

Molecular modeling of the Abeta1-42 peptide from Alzheimer's disease

The three-dimensional structure of the Alzheimer's disease Abeta1-42 peptide was predicted by sequence homology, threading approaches and by experimental observations. The Abeta molecule displayed a Greek key motif with four antiparallel beta-strands. To shield thermodynamically unfavorable domains, two Abeta molecules interact with each other to generate a beta-barrel structure with a hydrophilic surface and a hydrophobic core. The N-terminal domains of the dimer form crevices into which the non-polar C-termini are accommodated to yield a globular structure 27x32 A in diameter. Alternatively, the C-terminal domains of two opposing dimers could be extended to form an antiparallel beta- sheet. The stacking of these building blocks generates a helical protofilament. To create a thermodynamically more favorable structure, three protofilaments associate into a right-handed triple helix with a hydrophobic beta-sheet completely surrounded by the hydrophilic beta- barrels made of residues 1-28. Two triple helical strands can further associate into a right-handed amyloid filament. Although our model did not meet all the expected criteria, it nevertheless exhibited a series of naturally disposed structural features, revealed by other biophysical studies utilizing synthetic Abeta peptides. These characteristics are of functional significance in terms of Abeta- topology, fibril formation and cytotoxicity. The model also suggests that Abeta may not exist in a thermodynamically stable conformation, but rather as an ensemble of metastable dimeric structures some of which are capable of generating an extended C-terminal antiparallel beta-sheet essential in the promotion of fibrillogenesis.      

Molecular modelling of the ORL1 receptor and its complex with nociceptin

The opioid receptor like (ORL1) receptor is a G-protein coupled receptor superfamily, and regulates a plethora of neurophysiological functions. The structural requirements for receptor activation by its endogenous agonist, nociceptin (FGGFTGARKSARKLANQ), differ markedly from those of the kappa-opioid receptor and its putative peptide agonist, dynorphin A (YGGFLRRIRPKLKWDNQ). In order to probe the functional architecture of the ORL1 receptor, a molecular model of the receptor has been built, including the TM domain and the extra- and intracellular loops. An extended binding site able to accommodate nociceptin-(1-13), the shortest fully active analogue of nociceptin, has been characterized. The N-terminal FGGF tetrapeptide is proposed to bind in a highly conserved region, comprising two distinct hydrophobic pockets in a cavity formed by TM helices 3, 5, 6 and 7, capped by the acidic second extracellular (EL2) loop controlling access to the TM elements of the peptide binding site. The nociceptin conformation provides for the selective preference of the ORL1 receptor for nociceptin over dynorphin A, conferred by residue positions 5 and 6 (TG versus LR), and the favourable interaction of its highly positively charged core (residues 8-13) with the EL2 loop, thought to mediate receptor activation. The functional roles of the EL2 loop and the conserved N-terminal tetrapeptide opioid 'message' binding site are discussed in the context of the different structural requirements of the ORL1 and kappa-opioid receptors for activation.      

Strategy and Effects of Polyproline Peptide Stapling by Copper(I)-Catalyzed Alkyne–Azide Cycloaddition Reaction

Polyproline is a unique type of peptide that has a stable, robust, and well-defined helical structure in an aqueous environment. These features have allowed polyproline to be used as a nanosized scaffold for applications in chemical biology and related fields. To understand its structural properties and to expand the applications, this secondary structure was tested systematically by stapling the peptide at different locations with staples of various lengths. Using the efficient copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC), we successfully prepared stapled polyproline and investigated the impact of this peptide macrocyclization through circular dichroism analysis. Whereas the stapling seems to have no significant effect on polyproline helix II (PPII) conformation in water, the location and the length of the staple affect the transformation of conformation in n-propanol. These results provide valuable information for future research using peptide stapling to manipulate polyproline conformation for various applications.     

A consensus residue analysis of loop and helix-capping residues in four- alpha-helical-bundle proteins

A statistical study was performed on a set of proteins which adopt the four-alpha-helical-bundle tertiary motif in order to determine amino acid occurrences at helix-capping and loop positions. Eight X-ray crystal structures from the Brookhaven Protein Data Bank (PDB) were examined and N", N', Ncap, Ccap, C' and C" residues were assigned. In addition, a set of 55 protein sequences for the analogous proteins from different strains and species was taken from the Protein Information Resource and Swiss-Prot databanks. The residues at the capping and loop positions in this expanded data set were deduced by aligning these sequences with those from the PDB files. Similar trends were observed in the two data sets. In general, polar residues were predominant in the loops, although aromatic residues were also fairly common. Glycine, a highly flexible residue with an excellent 'helix-breaking' ability, was very common at the Ccap, C' and C" residues. Proline, which can force sharp turns in the direction of a peptide backbone, was only common at the N" residue. Residues which can participate in the N- capping box motif were found with high frequency. Capping motifs at the helix C-termini (Schellman and alphaL motifs) were also somewhat common, while another helix N-terminal stabilizing motif, the hydrophobic stable, was not common. The data presented in this study should prove useful for applying the 'consensus residue' approach to the de novo design of loop regions in helical bundle proteins.      

Residual Helicity at the Active Site of the Histidine Phosphocarrier,HPr, Modulates Binding Affinity to Its Natural Partners

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr^9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr^9–30 is mainly disordered. We attempted to improve the affinity of HPr^9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr^9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.     

Site-directed mutagenesis of the hinge peptide from the hemagglutinin protein: enhancement of the pH-responsive conformational change

Environmentally responsive proteins and peptides are increasingly finding utility in various engineered systems due to their ability to respond to the presentation of external stimuli. A classic example of this behavior is the influenza hemagglutinin (HA) fusion protein. At neutral pH, HA exists in a non-fusogenic state, but upon exposure to low pH, the conformation of the structure changes to expose a fusogenic peptide. During this structural change, massive rearrangements occur in a subunit of HA (HA2). Crystallography data has shown that a loop of 28 amino acids (residues 54-81) undergoes a dramatic transition from a random coil to an alpha-helix. This segment connects to two flanking helical regions (short and long) to form a long, continuous helix. Here, we report the results of site-directed mutagenesis study on LOOP-36 to further understand the mechanism of this important stimulus-responsive peptide. The conformational transition of a bacterially expressed LOOP-36 was found to be less dramatic than has been previously reported. The systematic mutation of glutamate and histidine residues in the peptide to glutamines (glutamine scanning) did not impact the conformational behavior of the peptide, but the substitution of the glycine residue at position 22 with alanine resulted in significant pH-responsive behavior. Therefore this mutant stimulus-responsive peptide may be more valuable for future protein engineering and bionanotechnology efforts.     

Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

The loop exchange mutant chymosm 155–164 rhizopuspepsinwas expressed in Trichoderma reesei and exported into the mediumto yield a correctly folded and active product. The biochemicalcharacterization and crystal structure determination at 2.5Å resolution confirm that the mutant enzyme adopts a nativefold. However, the conformation of the mutated loop is unlikethat in native rhizopuspepsin and involves the chelation ofa water molecule in the loop. Kinetic analysis using two syntheticpeptide substrates (six and 15 residues long) and the naturalsubstrate, milk, revealed a reduction in the activity of themutant enzyme with respect to the native when acting on boththe long peptide substrate and milk. This may be a consequenceof the different charge distribution of the mutated loop, itsincreased size and/or its different conformation.     

An in vitro peptide folding model suggests the presence of the molten globule state during nascent peptide folding

Although molten globules have been widely accepted as a generalintermediate in protein folding, there is no clear evidenceto show their presence during nascent peptide folding. Thispaper concentrates on whether the molten globule state occurs,and if it does, when does it form during nascent peptide folding,by comparing the changes in conformation during peptide chainextension of staphylococcal nuclease R. The results show thata large N-terminal fragment of staphylococcal nuclease, SNR121,which already contains more than 80% amino acid sequence ofthe nuclease, is found to fulfill all the criteria for the moltenglobule state, suggesting that the molten globule should occurat a later stage of peptide elongation. At this stage the hydrophobiccollapse of the polypeptide chain occurs driven by the hydrophobicforce, which leads to the formation of a solvent-accessiblenon-polar core, characterized by the high ANS-binding fluorescence.The nascent peptide folding of the nuclease is a hierarchicalprocess that at the very least includes the following steps:secondary structure accumulation, pre-molten globule state,molten globule state, post-molten globule state and finallythe native state. Constant conformation adjustment is necessaryfor correct folding and active expression of the protein.     

Approaches to Introduce Helical Structure in Cysteine-Containing Peptides with a Bimane Group

An i−i+4 or i−i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i−i+4 constrained peptide adopts a 3₁₀-helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.