A Specific Protein Disorder Catalyser of HIV-1 Nef

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Authors

Adrien Lugari1, Sebastian Breuer2, Thibault Coursindel3, Sandrine Opi4,5,6, Audrey Restouin4,5,6, Xiaoli Shi1,7, Alexis Nazabal8, Bruce E. Torbett2, Jean Martinez3, Yves Collette4,5,6, Isabelle Parrot3, Stefan T. Arold9, and Xavier Morelli1

Organizations

  1. IMR Laboratory, CNRS-UPR 3243, Centre National de la Recherche Scientifique, Institut de Microbiologie de la Méditerranée (IMM) and Aix-Marseille Universités, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
  2. Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
  3. Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Université Montpellier 1–Université Montpellier 2, CC17-03, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France
  4. INSERM U891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France
  5. Institut Paoli-Calmettes, Marseille F-13009, France
  6. Université de la Méditerranée, Marseille F-13007, France
  7. State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 155 Yang Qiao Xi Lu, Fuzhou, Fujian 350002, China
  8. CovalX AG, Grabenstrasse 11A, Schlieren CH-8952, Switzerland
  9. Department of Biochemistry and Molecular Biology, Unit 1000, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009, USA

Abstract

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1–infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC50 of ∼16 μM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14–bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors.

CovalX Technology Used (Click each option to learn more)

HM2

K100

Outcomes

The HIV-1 Nef core domain (residues 56-206) and the Hck SH3 domain were expressed in E. coli BL21 DE3 as GST fusion proteins and then purified. The purified Nef (5 μM, 4.5 μL) was mixed with DLC27 or DLC27-14 before being diluted in 100% DMSO to a variety of concentrations (50 μM, 100 μM, 150 μM, 250 μM, 300 μM, 350 μM, 450 μM, 500 μM, 5 nM) and incubated for an hour at room temperature. The mixture was mixed with 5 μL of SH3-Hck (5 μM) and incubated again for an hour at room temperature giving the final protein concentrations of 2.5 μM Nef and 2.5 μM SH3-Hck. The samples were then cross-linked using the CovalX K100 Stabilization Kit by mixing 1 μL of the cross-linking reagent into each solution and then incubating them for an hour. 1 μL of the cross-linked mixture was mixed with the matrix (recrystallized sinapic acid (10 mg-1) in acetonitrile/water (1:1,v/v), TFA 0.1%) and 1 μL of the final mixture was spotted onto a MALDI plate. The plate was allowed to crystallize at room temperature before being measured by a mass spectrometer that had been modified with a CovalX HM2 detection system.

Source

10.1016/j.bmc.2011.10.051

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