Major Histocompatibility Complex Class I (FLA-E*01801) Molecular Structure in Domestic Cats Demonstrates Species-Specific Characteristics in Presenting Viral Antigen Peptides

Abstract

Feline immunodeficiency virus (FIV) infection in domestic cats is the smallest usable natural model for lentiviral infection studies. FLA-E*01801 was applied to FIV AIDS vaccine research. We determined the crystal structure of FLA-E*01801 complexed with a peptide derived from FIV (gag positions 40 to 48; RMANVSTGR [RMA9]). The A pocket of the FLA-E*01801 complex plays a valuable restrictive role in peptide binding. Mutation experiments and circular-dichroism (CD) spectroscopy revealed that peptides with Asp at the first position (P1) could not bind to FLA-E*01801. The crystal structure and refolding of the mutant FLA-E*01801 complex demonstrated that Glu and Trp in the A pocket play important roles in restricting P1D. The B pocket of the FLA-E*01801 complex accommodates M/T/A/V/I/L/S residues, whereas the negatively charged F pocket prefers R/K residues. Based on the peptide binding motif, 125 FLA-E*01801-restricted FIV nonapeptides (San Diego isolate) were identified. Our results provide the structural basis for peptide presentation by the FLA-E*01801 molecule, especially A pocket restriction on peptide binding, and identify the potential cytotoxic T lymphocyte (CTL) epitope peptides of FIV presented by FLA-E*01801. These results will benefit both the reasonable design of FLA-E*01801-restricted CTL epitopes and the further development of the AIDS vaccine. Feline immunodeficiency virus (FIV) is a viral pathogen in cats, and this infection is the smallest usable natural model for lentivirus infection studies. To examine how FLA I presents FIV epitope peptides, we crystallized and solved the first classic feline major histocompatibility complex class I (MHC-I) molecular structure. Surprisingly, pocket A restricts peptide binding. Trp blocks the left side of pocket A, causing P1D to conflict with Glu We also identified the FLA-E*01801 binding motif X (except D)-(M/T/A/V/I/L/S)-X-X-X-X-X-X-(R/K) based on structural and biochemical experiments. We identified 125 FLA-E*01801-restricted nonapeptides from FIV. These results are valuable for developing peptide-based FIV and human immunodeficiency virus (HIV) vaccines and for studying how MHC-I molecules present peptides.

Structure and Peptidome of the Bat MHC Class I Molecule Reveal a Novel Mechanism Leading to High-Affinity Peptide Binding

“…XP_006920478.1) were codon optimized and synthesized (GENEWIZ, Beijing). Then, they were built into pET-21a (Novagen) and named pET-21a-PtalH and pET-21a-PtalB, respectively (33). The constructed plasmids were transformed into Escherichia coli strain BL21 (DE3).…”

“…The constructed plasmids were transformed into Escherichia coli strain BL21 (DE3). After prokaryotic expression and purification as inclusion bodies, as described previously (33), the purified proteins were dissolved in 6 M guanidine hydrochloride at a final concentration of 30 mg/ml and stored at 220˚C.…”

“…At a molar ratio of P. alecto MHC I HC (Ptal-N*01:01): P. alecto MHC I L chain (Ptal-b2m): peptide (HEV-1, HEV-2, and HEV-1 PVL ) = 1: 1: 3, inclusion bodies were renatured using a previously described gradual dilution method in refolding buffer composed of 100 mM Tris (pH 8), 400 mM L-arginine, 2 mM EDTA, 5 mM reduced glutathione, and 0.5 mM glutathione disulfide for 24 h at 4˚C (33). Then, the product was concentrated with a 10-kDa filter followed by column chromatography (Superdex 200 16/60 column and Resource Q anion exchange column, GE Healthcare) to obtain the purified p/Ptal-N*01:01 complexes (33).…”

“…The product was termed Ptal-N*01:01 DMDL and was refolded with b2m and the peptide. The complex was then further purified by gel filtration and anion exchange chromatography as described previously (33).…”

“…Notably, Asn 66 in other mammals is usually a glutamic acid, which might create charge repulsion with Asp at P1 (33). Residue 66 in bats was mostly N/S/Q, side chains without marked charge properties, which might contribute to the stability of the charged amino acids at P1 and to the formation of a salt-bridge chain.…”