title: Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion.
abstract: The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America.
authors: Zhen
authors: Z
authors: Gong
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America.
authors: Jose M
authors: JM
authors: Martin-Garcia
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5401, United States of America.
authors: Sasha M
authors: SM
authors: Daskalova
authors: Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center of Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5901, United States of America.
authors: Felicia M
authors: FM
authors: Craciunescu
authors: Virginia G. Piper Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-6401, United States of America.
authors: Lusheng
authors: L
authors: Song
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America.
authors: Katerina
authors: K
authors: Dörner
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center of Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5901, United States of America.
authors: Debra T
authors: DT
authors: Hansen
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America.
authors: Jay-How
authors: JH
authors: Yang
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Virginia G. Piper Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-6401, United States of America.
authors: Joshua
authors: J
authors: LaBaer
authors: Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5401, United States of America; School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, United States of America.
authors: Brenda G
authors: BG
authors: Hogue
authors: Center for Infectious Diseases and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5401, United States of America; School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, United States of America.
authors: Tsafrir S
authors: TS
authors: Mor
authors: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, United States of America; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-1604, United States of America.
authors: Petra
authors: P
authors: Fromme
mesh_terms: D016915:AIDS Vaccines / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000276:immunology; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000276:immunology; D015497:HIV-1 / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000276:immunology; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000276:immunology; D020349:Surface Plasmon Resonance; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification
chemical_list: D016915:AIDS Vaccines; D029968:Escherichia coli Proteins; D015700:HIV Envelope Protein gp41; C545841:HIV-1 envelope protein gp41 (649-684); C461619:MalE protein, E coli; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; D014760:Viral Fusion Proteins