Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.
We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.
Center
R J
RJ
St. Vincent's Institute of Medical Research, Fitzroy, Australia.
Kobe
B
B
Wilson
K A
KA
Teh
T
T
Howlett
G J
GJ
Kemp
B E
BE
Poumbourios
P
P
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000688:Amylose; D002352:Carrier Proteins / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D003460:Crystallization; D018360:Crystallography, X-Ray; D015485:Deltaretrovirus Antigens / Q000096:biosynthesis / Q000737:chemistry*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015686:Gene Products, env / Q000096:biosynthesis / Q000737:chemistry*; D058619:Maltose-Binding Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D015682:Retroviridae Proteins, Oncogenic / Q000096:biosynthesis / Q000737:chemistry*; D054299:env Gene Products, Human Immunodeficiency Virus
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D015485:Deltaretrovirus Antigens; D029968:Escherichia coli Proteins; D015686:Gene Products, env; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D054299:env Gene Products, Human Immunodeficiency Virus; C055265:gp21 protein, Human T-lymphotropic virus 1; C461599:maltose transport system, E coli; D000688:Amylose