Tài liệu Báo cáo khoa học: NMR structural characterization of HIV-1 virus protein U cytoplasmic

NMR structural characterization of HIV-1 virus protein U

cytoplasmic domain in the presence of

dodecylphosphatidylcholine micelles

Marc Wittlich1,2, Bernd W. Koenig1,2, Matthias Stoldt1,2, Holger Schmidt1,2,* and Dieter Willbold1,2

1 Institut fu¨r Strukturbiologie und Biophysik (ISB-3), Forschungszentrum Ju¨lich, Germany

2 Institut fu¨r Physikalische Biologie, Heinrich-Heine-Universita¨t Du¨ sseldorf, Germany

Introduction

VpU (virus protein U) is an 81 amino acid transmem￾brane protein encoded by HIV-1 and some simian

immunodeficiency virus strains, e.g. SIVCPZ. VpU is

not essential for virus replication in cell culture, and is

thus often called accessory protein.

The most well-defined function of VpU is downregu￾lation of CD4 in the endoplasmic reticulum, which is

mediated by the cytoplasmic region of the protein [1,2].

This function involves binding and recruitment of the

b-transducin repeat-containing protein (b-TrCP) [3,4]

Keywords

CD4; DPC micelle; HIV-1 VpU; NMR;

solution structure

Correspondence

D. Willbold, Forschungszentrum Ju¨lich

GmbH, ISB-3, 52425 Ju¨lich, Germany

Fax: +49 2461612023

Tel: +49 2461612100

E-mail: [email protected]

*Present address

Max-Planck-Institute for Biophysical

Chemistry, NMR-based Structural Biology,

Go¨ttingen, Germany

Database

Resonance assignment tables have been

deposited at the Biological Magnetic

Resonance Data Bank (BMRB) under the

accession code 15513

(Received 31 May 2009, revised 2

September 2009, accepted 7 September

2009)

doi:10.1111/j.1742-4658.2009.07363.x

The HIV-1 encoded virus protein U (VpU) is required for efficient viral

release from human host cells and for induction of CD4 degradation in the

endoplasmic reticulum. The cytoplasmic domain of the membrane protein

VpU (VpUcyt) is essential for the latter activity. The structure and dynam￾ics of VpUcyt were characterized in the presence of membrane simulating

dodecylphosphatidylcholine (DPC) micelles by high-resolution liquid state

NMR. VpUcyt is unstructured in aqueous buffer. The addition of DPC

micelles induces a well-defined membrane proximal a-helix (residues I39–

E48) and an additional helical segment (residues L64–R70). A tight loop

(L73–V78) is observed close to the C-terminus, whereas the interhelical lin￾ker (R49–E63) remains highly flexible. A 3D structure of VpUcyt in the

presence of DPC micelles was calculated from a large set of proton–proton

distance constraints. The topology of micelle-associated VpUcyt was

derived from paramagnetic relaxation enhancement of protein nuclear spins

after the introduction of paramagnetic probes into the interior of the

micelle or the aqueous buffer. Qualitative analysis of secondary chemical

shift and paramagnetic relaxation enhancement data in conjunction with

dynamic information from heteronuclear NOEs and structural insight from

homonuclear NOE-based distance constraints indicated that micelle-associ￾ated VpUcyt retains a substantial degree of structural flexibility.

Abbreviations

DHPC, dihexanoyl phosphatidylcholine; DPC, dodecylphosphatidylcholine; DPC-d38, perdeuterated DPC; HSQC, heteronuclear single

quantum coherence; PRE, paramagnetic relaxation enhancement; TFE, trifluoroethanol; VpU, virus protein U; VpUcyt, C-terminal,

cytoplasmic domain of VpU (residues 39–81) plus N-terminal Gly-Ser dipeptide; b-TrCP, b-transducin repeat-containing protein; TASK,

TWIK-related acid-sensitive K+ channel; TWIK, tandem of P domains in a weak inwardly rectifying K+ channel.

6560 FEBS Journal 276 (2009) 6560–6575 ª 2009 The Authors Journal compilation ª 2009 FEBS