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. 2009 Dec 16;131(49):17972-9.
doi: 10.1021/ja908170s.

NMR structure in a membrane environment reveals putative amyloidogenic regions of the SEVI precursor peptide PAP(248-286)

Ravi P R Nanga  1 , Jeffrey R Brender, Subramanian Vivekanandan, Nataliya Popovych, Ayyalusamy Ramamoorthy
Affiliations

NMR structure in a membrane environment reveals putative amyloidogenic regions of the SEVI precursor peptide PAP(248-286)

Ravi P R Nanga et al. J Am Chem Soc. .

Abstract

Semen is the main vector for HIV transmission worldwide. Recently, a peptide fragment (PAP(248-286)) has been isolated from seminal fluid that dramatically enhances HIV infectivity by up to 4-5 orders of magnitude. PAP(248-286) appears to enhance HIV infection by forming amyloid fibers known as SEVI, which are believed to enhance the attachment of the virus by bridging interactions between virion and host-cell membranes. We have solved the atomic-level resolution structure of the SEVI precursor PAP(248-286) using NMR spectroscopy in SDS micelles, which serve as a model membrane system. PAP(248-286), which does not disrupt membranes like most amyloid proteins, binds superficially to the surface of the micelle, in contrast to other membrane-disruptive amyloid peptides that generally penetrate into the core of the membrane. The structure of PAP(248-286) is unlike most amyloid peptides in that PAP(248-286) is mostly disordered when bound to the surface of the micelle, as opposed to the alpha-helical structures typically found of most amyloid proteins. The highly disordered nature of the SEVI peptide may explain the unique ability of SEVI amyloid fibers to enhance HIV infection as partially disordered amyloid fibers will have a greater capture radius for the virus than compact amyloid fibers. Two regions of nascent structure (an alpha-helix from V262-H270 and a dynamic alpha/3(10) helix from S279-L283) match the prediction of highly amyloidogenic sequences and may serve as nuclei for aggregation and amyloid fibril formation. The structure presented here can be used for the rational design of mutagenesis studies on SEVI amyloid formation and viral infection enhancement.

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Figures

Figure 1
Figure 1
The finger print region of a 2D 1H-1H NOESY spectrum of SDS micelles containing PAP248–86 showing NOE connectivities among Hα nuclei.
Figure 2
Figure 2
NOE intensity plot for the amino acid residues of PAP248–286 showing the NOE connectivity among residues. Thicker lines correspond to stronger NOE intensities.
Figure 3
Figure 3
Alpha proton chemical shift index (CSI) for PAP248–286 showing the disordered N-terminal end (CSI near zero) and the central and C-terminal helical regions (CSI <−0.1). The Ha chemical shift of K272 was not detected (red). The CSI was calculated by subtracting the Ha chemical shifts values measured for the peptide from the random coil shifts values for the respective amino acid reported in the literature.
Figure 4
Figure 4
Histogram of the number of NOEs detected versus the residue number showing the number of intra-residue, sequential (i – j = 1), and medium range (i – j = 2, 3, 4) NOEs detected. Long-range (i – j>4) NOEs were not observed.
Figure 5
Figure 5
(A) Secondary structure representation of an overlaid ensemble of NMR-derived conformers for PAP248–286 showing the helical region from V262-H270, the highly flexible N- and C-termini, and the short helix from S279-L283. (B) Secondary structure representation of PAP248–286 extracted from the crystal structure of human PAP. In the crystal structure, residues L283-Y286 are in a β-sheet conformation, while residues K251-I277 are in a helical conformation with a distortion at G261. (C) All atom representation of the PAP248–286 conformational ensemble.
Figure 6
Figure 6
Ramachandran plot showing the Phi and Psi angles for the ensemble of conformers of the PAP248–286 peptide.
Figure 7
Figure 7
Finger print region of a 2D 1H-1H TOCSY spectrum of SDS micelles containing PAP248–286 after the addition of 0.8 mM MnCl2. The complete quenching of almost all peaks indicates that the peptide is well exposed to the solvent and does not penetrate deeply into the micelle.
Figure 8
Figure 8
Prediction of the amyloidogenic propensity of the PAP248–286 sequence by AGGRESCAN. Red, orange, and grey colors indicate regions of high, moderate, and low amyloidogenic propensity respectively.
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