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 MPRTM 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 MBPAAA-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.
| Original language | English |
|---|---|
| Article number | e0136507 |
| Journal | PLoS One |
| Volume | 10 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - Aug 21 2015 |
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ASJC Scopus subject areas
- Agricultural and Biological Sciences(all)
- Biochemistry, Genetics and Molecular Biology(all)
- Medicine(all)
Cite this
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. / Gong, Zhen; Martin-Garcia, Jose M.; Daskalova, Sasha M.; Craciunescu, Felicia M.; Song, Lusheng; Dörner, Katerina; Hansen, Debra T.; Yang, Jay How; Labaer, Joshua; Hogue, Brenda G.; Mor, Tsafrir S.; Fromme, Petra.
In: PLoS One, Vol. 10, No. 8, e0136507, 21.08.2015.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Biophysical characterization of a vaccine candidate against HIV-1
T2 - The transmembrane and membrane proximal domains of HIV-1 gp41 as a maltose binding protein fusion
AU - Gong, Zhen
AU - Martin-Garcia, Jose M.
AU - Daskalova, Sasha M.
AU - Craciunescu, Felicia M.
AU - Song, Lusheng
AU - Dörner, Katerina
AU - Hansen, Debra T.
AU - Yang, Jay How
AU - Labaer, Joshua
AU - Hogue, Brenda G.
AU - Mor, Tsafrir S.
AU - Fromme, Petra
PY - 2015/8/21
Y1 - 2015/8/21
N2 - 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 MPRTM 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 MBPAAA-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.
AB - 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 MPRTM 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 MBPAAA-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.
UR - http://www.scopus.com/inward/record.url?scp=84942884607&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84942884607&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0136507
DO - 10.1371/journal.pone.0136507
M3 - Article
C2 - 26295457
AN - SCOPUS:84942884607
VL - 10
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 8
M1 - e0136507
ER -