Solution Structure of the Human Immunodeficiency Virus Type 1 p6 Protein

Torgils Fossen,Victor Wray,Karsten Bruns,Judhi Rachmat,Peter Henklein,Uwe Tessmer,Annette Maczurek,Patricia Klinger,Ulrich Schubert

doi:10.1074/jbc.m507375200

Abstract

The human immunodeficiency virus type 1 p6 protein represents a docking site for several cellular and viral binding factors and fulfills major roles in the formation of infectious viruses. To date, however, the structure of this 52-amino acid protein, by far the smallest lentiviral protein known, either in its mature form as free p6 or as the C-terminal part of the Pr55 Gag polyprotein has not been unraveled. We have explored the high resolution structure and folding of p6 by CD and NMR spectroscopy. Under membranous solution conditions, p6 can adopt a helix-flexible helix structure; a short helix-1 (amino acids 14-18) is connected to a pronounced helix-2 (amino acids 33-44) by a flexible hinge region. Thus, p6 can be subdivided into two distinct structural and functional domains; helix-2 perfectly defines the region that binds to the virus budding factor AIP-1/ALIX, indicating that this structure is required for interaction with the endosomal sorting complex required for transport. The PTAP motif at the N terminus, comprising the primary late assembly domain, which is crucial for interaction with another cellular budding factor, Tsg101, does not exhibit secondary structure. However, the adjacent helix-1 may play an indirect role in the specific complex formation between p6 and the binding groove in Tsg101. Moreover, binding studies by NMR demonstrate that helix-2, which also comprises the LXXLF motif required for incorporation of the human immunodeficiency virus type 1 accessory protein Vpr into budding virions, specifically interacts with the Vpr binding region, indicating that under the specific solution conditions used for structure analysis, p6 adopted a functional conformation.

Highlights

The plasma membrane targeting of Gag and lines the inner shell of the mature virus particle, capsid forms the conical core shell encasing NC, and NC regulates packaging and condensation of the viral genome (1– 6)
2 The abbreviations used are: HIV-1, human immunodeficiency virus-1; ESCRT, endosomal sorting complex required for transport; NC, nucleocapsid; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser enhancement spectroscopy; TFE, trifluoroethanol; TOCSY, total correlation spectroscopy; Solid-phase peptide synthesis (SPPS), solid phase peptide synthesis; sp6, synthetic p6 Gag protein; UEV, ubiquitin E2 variant sequence; L-domain, late assembly domain; HPLC, high pressure liquid chromatography; r.m.s., root mean square; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; E2, ubiquitin conjugating enzyme
We have developed a first insight into the molecular structure of the HIV-1 p6 Gag protein

Summary

EXPERIMENTAL PROCEDURES

Peptide Synthesis, Purification, and Characterization—Peptide synthesis were performed using the Fmoc (N-(9-fluorenyl)methoxycarbonyl)/t-butyl strategy on an Applied Biosystems automated Pioneer peptide synthesizer (Applied Biosystems Darmstadt) using 0.5 g of TentaGel SAC Gln(Trt) resin (0.18 mmol/g; Rapp Polymere Tubingen), similar to a protocol described previously by us for the synthesis of the 96-residue HIV-1 regulator protein Vpr (35). Binding Studies of sp6-(1–52) and sVpr-(26 –33)—After recording one- and two-dimensional NMR spectra of sp6-(1–52), the same 50% TFE NMR sample as above was transferred to a new vial containing an equimolar amount of the peptide sVpr-(26 –33) (LKSEAVRH), comprising the domain of the HIV-1NL4–3 Vpr that was shown previously to bind to p6. After complete dissolution of sVpr-(26 –33), the solution was transferred back into the NMR sample tube, and identical NMR experiments were performed under exactly the same conditions as for sp6-(1–52) at 300 K. All NMR experiments with homogenous sp6-(1–52) and heterogeneous sp6-(1–52) mixed with sVpr-(26 –33) at ratios of 1:1 or 1:2 were performed under identical conditions In these solutions, the p6 resonances were resolved from those of the sVpr-(26 –33) peptide, which was unambiguously assigned at both concentrations (see supplemental Fig. 2 and Table 6).

TABLE ONE

RESULTS

TABLE TWO

DISCUSSION