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Structure-related roles for the conservation of the HIV-1 fusion peptide sequence revealed by NMR Soraya Serrano, Nerea Huarte, Edurne Rujas, David Andreu, Jose L. Nieva, and M. Angeles Jimenez Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00745 • Publication Date (Web): 20 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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Biochemistry

STRUCTURE-RELATED ROLES FOR THE CONSERVATION OF THE HIV-1 FUSION PEPTIDE SEQUENCE REVEALED BY NMR Soraya Serrano¶, Nerea Huarte§, Edurne Rujas§, David Andreu‡, José L. Nieva§, and María Angeles Jiménez¶ * ¶

Institute of Physical Chemistry “Rocasolano” (IQFR-CSIC), Serrano 119, E-28006 Madrid, Spain

§

Biofisika Institute (CSIC-UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain. ‡

Proteomics and Protein Chemistry Unit, Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Dr. Aiguader 88, 08003 Barcelona, Spain.

*To whom correspondence should be addressed. E-mail: [email protected] KEYWORDS: NMR spectroscopy; peptide conformation; HIV fusion peptide; oligomerization.

ACS Paragon Plus Environment

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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 tripeptide FLGFLG is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the 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 the C-terminus 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 lowpolarity medium 25 % 1,1,1,3,3,3-hexafluoro-2-propanol (v/v; HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas the wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure when 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.


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INTRODUCTION HIV infection causes a malfunctioning of the immune system known as acquired immunodeficiency syndrome (AIDS). The process initiates with fusion of virus and host cell membranes, promoted by the Env glycoprotein, which is composed of the gp120 and gp41 subunits (reviewed in: (1-3)). First the surface subunit gp120 binds to cell receptors and coreceptors on T lymphocytes. This binding promotes a conformational change in the Env complex leading to the formation of the intermediate pre-hairpin state. In this pre-hairpin state the gp41 subunit, which consists of ectodomain, transmembrane region and cytoplasmic tail (Figure 1), inserts into the target cell membrane through the N-terminal fusion peptide (FP), while still anchored to the viral membrane by the transmembrane domain region. The pre-hairpin subsequently collapses and folds back onto itself giving rise to a bundle of 6 helices (6-HB, also envisioned as a trimer of hairpins), where the three C-terminal helices (CHR region of gp41; Figure 1), are packed in a direction opposite to the external hydrophobic groove formed by the N-terminal helices (NHR region of gp41; Figure 1)(1, 4). When the 6-helix bundle is formed, the NHR and CHR regions interact with each other, whereby the two membranes approach and merge. It is assumed that the conformational energy released after the formation of the 6-HB is used to bring the membranes closer and induce fusion (3). Mutagenesis studies confirmed that the FP region (residues 512-524, numbered as in the Env complex of the prototypic HXBc2 isolate; Figure 1) plays a crucial role in the fusion process (58). The FP sequence is rich in Gly, Leu and Phe, which make it a conformationally flexible, hydrophobic region. Sustaining this notion, site-directed mutagenesis revealed that Gly521 and Gly524 were critical for fusion activity, while the replacement of Phe522 by Gly inhibited the process (9, 10). The total conservation of the FLGFLG tandem repeat further suggests that,


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beyond preservatation of hydrophobicity, sequence order is also an important feature (5). Herein we use NMR to address the relevance of maintaining the order of those residues for attaining the membrane-bound structure of the FP region.

Figure 1: Sequences of the wtFP-tag and scrFP-tag peptides. The residues derived from the FP region are indicated in bold, with the residues of the central hydrophobic region of FP underlined. The residues corresponding to the 2F5 epitope of the MPER region are shown in plain and the connecting region in italics. Ahx is the 6-aminohexanoic acid connecting unit. In the upper part, all domains of the ectodomain of the gp41 protein are schematically displayed (FP, fusion peptide; PR, fusion peptide proximal region; NHR, N-terminal heptad repeat; ID, immunodominant loop; CHR, C-terminal heptad region; MPER, Trprich region containing epitopes 2F5 and 4E10; (11), as well as the transmembrane domain (TMD) and cytoplasmic tail (CT). A few residues are numbered at the top of wtFP-tag sequence to show their correspondence to native Env glycoprotein (11).

To date, two three-dimensional NMR structures adopted by the FP region of gp41 in membrane mimics are available at the Protein Data Bank. The first deposited structure was determined in SDS micelles (PDB code: 2ARI) and corresponds to a 37-mer peptide spanning residues 512-541 of gp41, which contains a non-native polar segment at its C-terminal end (12). The other structure was determined in DPC micelles (PDB code: 2PJV) for a peptide corresponding to residues 512-535 of gp41, which also contains a C-terminal extension of Lys amino acids to increase solubility (13). In both cases, solution NMR revealed the adoption of


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mainly α-helix structure. Therefore, it was inferred that the helical structure represents a relevant conformation adopted by the FP region under physiological conditions. In this work we make use of peptides that combine through a flexible connection the FP sequence of HIV-1 with an epitope that allows immunological detection upon membrane insertion (Figure 1). The resolution of their structure by NMR in nonpolar media demonstrates the total absence of interactions between the two components of the peptides, which allows the FP structure to be analyzed independently. The data further indicate that altering the order of core residues LFLGFLG, while keeping the overall hydrophobicity of the peptide, has an impact on the secondary structure adopted by the FP in the membrane environment, which results in their inability to homo-oligomerize therein. We conclude that the correct order of the FLGFLG dipeptide sequence sustains FP insertion into membranes as helical trimers, explaining in part its conservation degree. MATERIALS AND METHODS Materials 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) was purchased from Avanti Polar Lipids (Birmingham, AL, USA). Dodecylphosphocholine (DPC) was from Anatrace (Maumee, OH, USA).

1,1,1,3,3,3-hexafluoroisopropanol-D2

(HFIP-D2;

98%

deuteration)

and

dodecylphosphocholine-D38 (DPC-D38, 98% deuteration) were from Cambridge Isotope Labs (Andover, MA, USA) Peptides The wtFP-tag and scrFP-tag peptides (sequences displayed in Figure 1) were synthesized in Cterminal carboxamide form by Fmoc solid-phase methods, purified by reverse phase HPLC (>95% homogeneity), and characterized by matrix-assisted time-of-flight (MALDI-TOF) mass


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spectrometry. Peptides were dissolved in dimethylsulfoxide (DMSO, spectroscopy grade) and their concentration determined by the biscinchoninic acid microassay (Pierce, Rockford, IL, USA). NMR spectroscopy Samples for NMR spectra acquisition at about 0.6-0.7 mM peptide concentration were prepared by dissolving the lyophilized peptide (approx. 0.9-1.0 mg) in 0.5 ml of a H2O/D2O (9:1 ratio by volume) solution containing 2 mM HEPES buffer at pH 6.8 and either 25 % HFIP-D2 (HFIP media) or 20 mM DPC-D38 (DPC micelles). All samples contained sodium 2,2-dimethyl2-silapentane-5-sulfonate (DSS) as internal reference for 1H chemical shifts. pH was measured with a glass micro electrode and not corrected for isotope effects. 1D and 2D NMR spectra were acquired on a Bruker Avance-600 spectrometer operating at a proton frequency of 600.13 MHz and equipped with a cryoprobe and processed using the standard TOPSPIN program (Bruker Biospin, Karlsruhe, Germany), as we previously described for other gp41-derived peptides (14). 1

1

H chemical shifts were assigned by analysing 2D [1H-1H]-COSY, [1H-1H]-TOCSY and [1H-

H]-NOESY with the aid of the SPARKY software (T.D. Goddard and D. G. Kneller, SPARKY

3, University of California, San Francisco, USA) and following the standard sequential strategy (15, 16). Based on the cross-peaks between the protons and the bound carbons observed in [1H13

C]-HSQC spectra, the

13

C chemical shifts could be assigned for the two peptides in the

presence of HFIP. However, they could not be assigned in the presence of DPC micelles, because they led, as commonly observed (14), to signal broadening, which, in its turn, decreased the signal-to-noise ratio of the low sensitive [1H-13C]-HSQC spectra acquired at natural

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C

abundance. Tables S1-S5 list the assigned chemical shifts, which have been deposited at


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BioMagResBank under the accession codes 34132 (wtFP-tag in HFIP), 34130 (wtFP-tag in DPC micelles), 34133 (scrFP-tag in HFIP), and 34134 (scrFP-tag in DPC micelles).

Table 1. Relevant structure calculation data: Statistics for the ensemble of the 20 lowest target function conformers of wtFP-tag and scrFP-tag in DPC micelles and in 25 % HFIP. PDB codes are indicated in parenthesis. wtFP-tag

scrFP-tag

HFIP

DPC

HFIP

DPC

(5NWU)

(5NVP)

(5NWV)

(5NWW)

Upper limit distances

277

411

304

286

Dihedral angles (ϕ, ψ)

60

60

60

57

7.3 ± 2.9

6.2 ± 2.3

6.9 ± 2.2

6.3 ± 2.0

(8.3 ± 2.6)

(6.9 ±2.3)

(8.1 ± 2.4)

(7.2 ± 2.2)

0.7 ± 0.2

0.8 ± 0.3

0.7 ± 0.3

0.8 ± 0.3

(1.5 ± 0.3)

(1.2 ±0.3)

(1.4 ±0.3)

(1.4 ± 0.4)

0.5 ± 0.1

0.5 ± 0.2

0.5 ± 0.2

0.9 ± 0.7

(1.3 ± 0.2)

(1.2 ±0.2)

(1.3 ± 0.2)

(1.8 ± 0.7)

No of restraints

Pairwise RMSD (Å) Backbone atoms (all heavy atoms) Full-length FP helix (517-528) a MPER-H1 helix (656-670) b

Ramachandran plot (%) Residues in most favoured regions

94.6

99.2

97.3

86.9

Residues in additional allowed regions

5.4

0.8

2.7

13.1

0

0

0

0

0

0

0

0

Residues in generously allowed regions Residues in disallowed regions a

Residues belonging to FP helix are 517-528 for wtFP-tag and scrFP-tag in HFIP, 515-528 for wtFP-tag in DPC, and 520-528 for scrFP-tag in DPC. b Residues belonging to MPER-H1 helix are 656-670 for the two peptides in both 25 % HFIP and DPC micelles.


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Structure calculation Structures were calculated for the two peptides in non-polar medium (25 % HFIP) and in the presence of detergent micelles (20 mM DPC). For each peptide and condition, distance constraints were obtained from the cross-peaks present in the corresponding 150 ms 2D [1H-1H]NOESY spectra and dihedral angle restraints for φ and ψ angles from chemical shifts using the TALOS program (17). Then, the standard iterative protocol for automatic NOE assignment of the program CYANA 2.1 (18), which comprises seven cycles of combined automated NOE assignment and structure calculation of 100 conformers per cycle (19), was applied. The final structure for each peptide and condition is the ensemble of the 20 conformers with lowest target function. These structures were visualised and examined using MOLMOL (20), and their quality assessed using PROCHECK/NMR (21) as implemented at the Protein Structure Validation Suite server (PSVS server: http://psvs-1_5-dev.nesg.org). They have been deposited at the PDB data bank with accession codes 5NWU (wtFP-tag in HFIP), 5NVP (wtFP-tag in DPC micelles), 5NWV (scrFP-tag in HFIP), and 5NWW (scrFP-tag in DPC micelles). Their structural statistics data are listed in Table 1, and in Supplementary Table S6. Peptide analyses by Western blot after vesicle flotation To analyze the oligomeric state of peptides after their association with membranes, large unilamellar vesicles were incubated with peptide at a 1:100 peptide-to-lipid mole ratio. The resulting vesicle-peptide complexes were subsequently floated in a sucrose gradient. After centrifugation at 436,000 × g for 3 h in a TLA 120.2 rotor (Beckman Coulter, Brea CA, USA), floating and pellet fractions were collected and subjected to electrophoretic analysis in a 10-20% Tricine gel (NOVEX Tricine gels, Invitrogen). For Western blot, peptides were transferred to nitrocellulose membranes and probed with antibody 2F5.


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RESULTS Peptide design To determine at the atomic level the structural effects of changing the order of the conserved FP core sequence, we designed two FP-derived peptides, named wtFP-tag and scrFP-tag (Figure 1). The wtFP-tag peptide contained the FP-derived sequence (514GIGALFLGFLGAAGS528) and the epitope 2F5 attached by a flexible spacer, consisting of two Lys and one 6-amino hexanoic acid, Ahx, residues. Residues from the FP region were selected taking into account the length required to sustain function (7). The scrFP-tag peptide had the same FP residues as wtFP-tag, (514GIGAFGLLGFLAAGS528), but the order in the conserved central sequence, where most of the hydrophobic residues Leu and Phe are found, was altered (underlined).


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Figure 2. Relevant NMR data. ∆δHα values (∆δHα = δHαobserved – δHαRC, ppm) plotted as a function of sequence for peptides wtFP tag (A) and scrFP-tag (B) in 25 % HFIP (black bars) and in the presence of DPC micelles (white bars) at pH 6.8 and 25 ºC. The dashed lines indicate the random coil (RC) range


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(|∆δHα| ≤ 0.05 ppm). δHαRC values were taken from (22). As in Figure 1, the residues derived from the FP region are indicated in bold, with the residues of the central hydrophobic region of FP underlined, and those at the linker in italics. “X” stands for Ahx. (C) 1H-1H NOESY spectral regions of the wtFP-tag peptide in 20 mM DPC at pH 6.8 and 25 °C. The non-sequential NOEs at the left panels are αN(i,i+2), αN(i,i+3) and αN(i,i+4), and those at the right panel corresponds to NOEs between side chain protons from aromatic and methyl groups. All of them are boxed; those from the MPER-tag region with a dashed line.

NMR structural study The two FP-derived peptides were subsequently analyzed by solution NMR in a non-polar (25 % HFIP) and in a membrane-mimetic media (20 mM DPC). An essentially complete assignment of 1H chemical shifts was achieved for the two peptides in both solvent conditions. Also,

13

C

chemical shifts could be assigned for the two peptides in the presence of HFIP, but not in DPC micelles (Tables S1-S5; see Materials and methods). Once assigned the chemical shifts, the finding that the 1Hα chemical shift deviations (∆δHα = ∆δHαobserved – ∆δHαRC, ppm; Figure 2A-B) for most residues lie outside the random coil range (22) evidences that the two peptides form ordered structures in both HFIP and DPC micelles conditions. The negative sign of these ∆δHα values shows that both the FP-derived segment and the MPER-tag region form helical structures, whereas their large magnitudes indicate that the helices are highly populated (23-25). In addition, the positive and large ∆δCα values (∆δCα = ∆δCαobserved – ∆δCαRC, ppm; Figures S1A-S2A) corroborate that the peptides are helical in the presence of HFIP. Further and stronger evidence for helix formation comes from the sets of NOEs, which contain numerous non-sequential NOEs characteristic of helices, such as those seen in Figure 2C for wtFP-tag in DPC micelles (see Figure S3 for wtFP-tag in HFIP, and for scrFP-tag in the two media; and also the NOE summaries in Figures S1-S2). To visualize the helical conformations adopted by the two peptides, we performed structure calculations based on the distance restrictions derived from the


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observed NOEs and from the dihedral angle restraints obtained from chemical shifts (Tables 1 and S6) and following the protocol described at the Materials and Methods section. Location of all the dihedral angles ϕ and ψ in favorable regions of the Ramachandran map supports the good quality of the resulting structures (Table 1). Nevertheless, considering all residues, the four structural ensembles look very poorly defined, since the pairwise RMSD values are very high (Table 1), as though the structures were completely disordered. However, the RMSD values for each of the regions considered separately (i.e., only FP, or solely MPER-tag) are quite low (Tables 1 and S6). This indicates that the structures of the two regions are well defined, but the linker is quite flexible, so that the orientation of one region relative to the other is not defined, as visualized in Figures 3 and S3. Thus, the two regions do not interact with each other under any of the experimental conditions examined, as further evidenced by the absence of NOEs between residues from the FP and MPER-tag regions. The chemical shift deviations determined in nonpolar media (see below) also agree with the two composing regions folding independently and non-interacting with each other in either of the two peptides. Therefore, we proceeded with a detailed analysis of the structure adopted by the FP region as an isolated entity.


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Figure 3. Ensembles of the 20 lowest target function structures calculated for peptides wtFP-tag (A) and scrFP-tag (B) in 25 % HFIP overlaid onto backbone atoms of FP region (top panels) and MPER-tag region (bottom panels). The FP region is displayed in green, the separator residues in gray, and the MPER-tag epitope in magenta.

Structure of the FP region: wild type versus scrambled sequence The structural ensembles of the FP-region are well defined in the two peptides in the two solvent media, as evidenced by the small RMSD values (Table 1; see also Figure S5). For peptide wtFP-tag in the presence of 25 % HFIP, the FP segment shows a well-defined regular αhelix that starts at Ala517 and extends up to Ser528, and even to the linker residues Lys529-


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Lys530 in many conformers (Figures 4A & S5A). The helix formed by peptide scrFP-tag in 25 % HFIP also comprises residues Ala517-Ser528/Lys530 (Figures 4C & S5C). Thus, despite their differences in sequence (Figure 1), the FP and scrFP helices in the non-polar solvent are quite similar, as seen in Figure 4E, and indicated by the low RMSD value for the backbone atoms (0.8 Å for residues 517-528). Evidently, the distribution of side chains around the helix axis is different (Figures 4A & 4C). By comparison, the FP and scrFP helices formed in the presence of DPC micelles exhibit several differences (Figures 4 & S5). Thus, the FP region in peptide wtFP-tag in DPC micelles forms an α-helix extending from Ile515 to Ala525, with residues 523-525/528 showing a 310 helix in some conformers of the structural ensemble (Figures 4B & S5B). In fact, the helix displays a distortion at residues Phe522-Leu523 (Figure 4B), which is also present in the helical structure of a longer FP-derived peptide in DPC micelles (Figure S6B) (12). In contrast, the helix adopted by peptide scrFP-tag in DPC micelles is markedly shorter than in the non-polar media. It spans from Leu520 to Ser528, with the preceding N-terminal region disordered (Figures 4D & S5D). Hence, the sequence differences between the scrFP and the wild type FP regions lead to distinct helical lengths in detergent micelles. The wild type FP helix is longer than the scrFP one (Figures 4F & S5).


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Figure 4. FP region in peptides wtFP-tag and scrFP-tag. A representative ribbon structure for the FP region in peptide wtFP-tag (green) in 25 % HFIP (A) and in 20 mM DPC (B), and for the scrFP region of peptide scrFP-tag (blue) in 25 % HFIP (C) and in 20 mM DPC (D). Side chains are displayed in magenta for non-polar residues (Ile, Leu, Ala), in yellow for aromatic residues (Phe), in cyan for Ser, and in blue for Lys. (E & F) Backbone atoms of the FP region of wtFP-tag (green) structure overlaid onto those of scrFPtag (blue) in 25 % HFIP (E) and in DPC micelles (F).


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Figure 5. Self-oligomerization in membranes determined by Western blot. (A) Lateral and top view of a representative ribbon structure of wtFP-tag (right) solved in DPC micelles displaying residues Gly514, Ala517, Gly521, Gly524 and Gly527 as green spheres, and of scrFP-tag (left) displaying residues Gly514, Ala517, Gly519, Gly522 and Gly527 as green spheres. As in Figure 4, side chains for non-polar


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residues (Ile, Leu, Ala) are displayed in magenta, for aromatic residues (Phe) in yellow, and for Ser in cyan. (B) Oligomerization state of peptides incubated with lipid vesicles, prior and after flotation in a sucrose gradient (‘input’ and ‘floated’ lanes, respectively). In both cases, the input peptide was recovered co-floating with vesicles, as evidenced by its absence in the pellet fraction.

FP-mediated oligomerization The FP helix formed by wtFP-tag peptide upon contact with DPC micelles exhibits a ridge where the Gly514, Ala517, Gly521, Gly524 and Gly527 residues are grouped (Figure 5A, left). In the regular α-helix formed in the non-polar solvent, these residues are also grouped, but the surface sinks at Gly521 (Figure S7). Likely, the continuous ridge is a consequence of the discontinuity observed at residues Phe522-Leu523 in the helix adopted in 20 mM DPC (Figure 4B). This arrangement of the Gly/Ala residues could facilitate the interaction between helices in a membrane environment (26). In contrast, the FP structure in the scrFP-tag peptide shows a certain degree of denaturation in DPC micelles, which leads to the loss of that surface (Figures 4D, 4F, 5A, right, and S5D). Even in the non-polar media where the scrFP-tag peptide forms a regular helix (Figure 4C), no continuous surface is found for the Gly residues, which is due to Gly521 and Gly524 in wtFP-tag occupying position 519 and 522 in scrFP-tag (Figure S7). Western blot (WB) analysis supported the possibility that the FP helix is prone to oligomerization in membranes, if the Gly/Ala edge is preserved (Figure 5). The MPER-tag region showed almost identical 1Hα chemical shift deviations for the peptides wtFP-tag and scrFP-tag in the two solvent conditions examined (HFIP and DPC micelles; Figures 2A-B and S8A-B). This fact evidences that the MPER-tag structure is mainly unaffected by the preceding sequence (see Supplementary material for further details), validating its usefulness for immunochemical detection of both peptides. The WB data revealed equal amounts of wtFP-tag monomers, dimers and trimers when incubated with vesicles (Figure 5B, left). In addition, the


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peptide remained associated with the vesicles after flotation indicating that the oligomeric forms existed in the membrane environment. This pattern of bands was not observed in the case of the scrFP-tag, even though this peptide also co-floated with the vesicles (Figure 5B, right). In this case, monomers were the dominant form, accompanied by a dim band corresponding to dimers, but trimers were not detected. DISCUSSION Viral FPs constitute a family of membrane-interacting hydrophobic domains, whose sequences are constrained for fulfilling several structure-function requirements, namely: i) establishment of native interactions required for folding within viral glycoprotein complexes prior to fusion; ii) insertion into the target cell membrane after fusion activation; and iii) refolding into membrane integral helical bundles hypothesized to emerge as end products of the fusion process (reviewed in: (27)). In the case of the HIV-1 Env glycoprotein it appears that dynamic fluctuations between closed and open pre-fusion states result in the transition of the FP between solvent-exposed and sequestered conformations, respectively (28, 29). It may be inferred that folding in the interior of the native gp120/gp41 complex will impose a greater degree of restriction on the amino acid sequence, while subsequent membrane interactions will depend to a greater extent on the degree of hydrophobicity. However, data in this work suggest that the degree of conservation of the HIV-1 FP can be due in part to the requirement of maintaining an oligomeric structure in membranes, once the fusion process is activated (model displayed in Figure 6). We have approached the structural relevance of conserving a correct residue order within the hydrophobic core of the FP by comparing the NMR structures adopted by wtFP-tag and scrFPtag in two non-polar media. According to the NMR data, the structures adopted by the FP region of the wtFP-tag peptide in the presence of 25% HFIP and in 20 mM DPC show structural


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differences (Figure 4A-B). In the two media, the structure is helical, but in HFIP it extends from Ala517 to Lys530, while in DPC it adopts an α-helix from Ile515 to Phe522, followed by a 310 helix from Leu523 to Ala525, with residues Phe522-Leu523 forming a rotation which causes a change in the direction of the α-helix relative to the helix 310. The rotation in Phe522-Leu523 may be due to the interactions of non-polar residues with the DPC micelles. As shown in Figure 4A-B, the side chains in the central area of the α-helix maintain practically the same arrangement in the two media, but the helices exhibit different curvatures. In the presence of HFIP, the FP helix is relatively straight, whereas the helix curves in DPC micelles, laying the aromatic residues Phe519 and Phe522 in a convex face and forming the Gly residues a ridge (Figures 4B and 5A). These environment-dependent structural differences suggest that the structure adopted by the FP region may be influenced by membrane composition, in agreement with previous studies by Jaroniec and coworkers (12). Thus, the structure resolved in HFIP for the wtFP-tag peptide (where the helix ranges from Ala517 to Lys530) is highly similar to that determined in SDS micelles for the peptide spanning residues 512-535 (12) (pdb code 2ARI; Figure S6), which shows an uninterrupted helix where the 12 residues of the Ile515-Ala526 segment are solvent-protected and micelle-embedded, while the back and front residues, Gly514 and Gly527, are located at the micellar interface. In contrast, the calculated DPC structure (where the helical region ranges from Ile515 to Ala525) resembles more closely the peptide structure containing residues 512-535 of gp41 also determined in DPC micelles (13) (pdb code 2PJV; Figure S6), which shows a helix of Ile515 to Ala525, but with a discontinuity at Leu520-Phe522 residues. The similarities found between the structures of the FP region in wtFP-tag peptide (residues 514-528) and in the longer peptides (2ARI and 2PJV) indicate that the N-terminal structure is mainly unaffected by the following sequence. In this


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sense, it is worth noting that the C-terminal regions of the longer peptides (residues 530-535 in 2PJV; and 531-541 in 2ARI) are disordered. Moreover, it is especially striking that the FP region of the scrFP-tag peptide in HFIP maintains the capacity to form a helical structure despite the alteration of the conserved central sequence of the FP (from LFLGFLG to FGLLGFL, Figure 1). In this medium, the length of the scrFP helix is approximately the same as that adopted by the FP region in the wtFP-tag peptide (Figure 4E). Obviously, the arrangement of the Phe and Leu side chains is different (Figures 4A & 4C). However, in the presence of 20 mM DPC, the helix formed by scrFP (Leu521-Ala526) is significantly shorter than that of FP in the wtFP-tag peptide (Ile515-Ala525) (Figures 4D & 4F). This shortening of the α-helix highlights the prominent role of the conserved residues at the center of the FP for determining its structural behavior. The latter would explain that the helical structure of the wtFP-tag FP region is less influenced by the medium than the scrFP region in srcFP-tag. Given these results, previous data indicating that even relatively conservative point mutations can have dramatic effects on fusion activity (9, 10, 30) could be explained by the specific importance of the interactions involving the mutated residue, and not only by the effect on the formation of the helical structure.


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Figure 6. Schematic representation showing the formation of the trimeric bundle of FP helices in membranes. (A) Upon fusion activation the FP (green loop) at the N-terminus of the gp41 NHR domain (gray cylinder) is propelled towards the target membrane. The FP loop is assumed to be flexible in just before insertion. (B) Different intermediates can precede and follow the formation of a helix trimer in membranes, which would be blocked by the alteration of the FP sequence.

The gathering of the Gly residues into a surface of the helix, conserved in both wtFP-tag structures, suggests that helix-helix interactions can ensue through these surfaces. Consistent with that idea we found trimeric forms, but not larger oligomers, in wtFP-tag membrane samples. These trimers were not observed in scrFP-tag samples, even though this peptide displayed a similar capacity for associating with vesicles. Thus, our data support self-oligomerization of FP helices in the membrane milieu as the structural feature that explains the disposition of hydrophobic residues in a correct order along the FP sequence. FP insertion into the target membrane as α-helices or extended structures has been proposed to embody successive intermediates during the HIV fusion process (31-34). As illustrated in Figure 6, the results in this


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work suggest that formation of an integral membrane trimeric helix bundle could constitute one among those intermediates. It is tempting to speculate that such sequence-dependent structure can help completing the 6HB in the membrane, as a final step of the fusion process (35). ACKNOWLEGMENTS This study was supported by the Spanish MINECO (CTQ2014-52633P to MAJ; BIO201129792 and BIO2015-64421-R to JLN; SAF2011-24899 and AGL2014-52395-C2-2-R to DA) and the Basque Government (IT838-13 to JLN). SS and ER received pre-doctoral fellowships from Spanish MINECO and the Basque Government, respectively. SUPPORTING INFO A short paragraph describing details on the structure of the MPER-tag region. NMR parameters (Figures S1 and S2), selected 1H-1H NOESY spectral regions (Figure S3), structural ensembles in DPC micelles (Figure S4), backbone structure of the FP region (Figure S5), comparison with the structures of other FP-derived peptides (Figure S6), and lateral and top view of representative ribbon structures (Figure S7) for peptides wtFP-tag and scrFP-tag. Comparison of the MPER-tag region in peptides wtFP-tag, scrFP-tag and full-length MPERp (Figure S8). 1H and

13

C chemical shifts (Tables S1-S5) and additional structural statistics data (Table S6) for

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