RESUMO
We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.
Assuntos
Adenina/química , Antineoplásicos/química , DNA Glicosilases/química , Inibidores de Integrase de HIV/química , Repetição Terminal Longa de HIV , Proteínas Inativadoras de Ribossomos Tipo 1/química , Antineoplásicos/farmacologia , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , DNA Glicosilases/farmacologia , DNA Viral/efeitos dos fármacos , DNA Viral/genética , Inibidores de Integrase de HIV/farmacologia , Humanos , Modelos Moleculares , Oligodesoxirribonucleotídeos/química , Conformação Proteica , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Relação Estrutura-Atividade , Integração Viral/efeitos dos fármacosRESUMO
We previously reported that lysozyme accounts for anti-HIV activity associated with the beta-core fraction of human chorionic gonadotropin [Lee-Huang, S., Huang, P. L., Sun, Y., Kung, H. F., Blithe, D. L. & Chen, H. C. (1999) Proc Natl Acad Sci U S A 96, 2678-81]. To define the structural and sequence requirements for anti-HIV activity, we carried out peptide fragmentation and activity mapping of human lysozyme. We identified two peptides that consist of 18 and 9 amino acids of human lysozyme (HL18 and HL9), corresponding to residues 98-115 and 107-115. HL18 and HL9 are potent inhibitors of HIV-1 infection and replication with EC(50)s of 50 to 55 nM, comparable to intact lysozyme. Scrambling the sequence or substitution of key arginine or tryptophan residues results in loss of antiviral activity. HL9, with the sequence RAWVAWRNR, is the smallest peptide we identified with full anti-HIV activity. It forms a pocket with its basic residues on the surface of the molecule. HL9 exists as an alpha-helix in native human lysozyme, in a region of the protein distinct from the muramidase catalytic site. Monte Carlo peptide folding energy minimizing simulation modeling and CD studies indicate that helical propensity does not correlate with antiviral activity. HL9 blocks HIV-1 viral entrance and replication, and modulates gene expression of HIV-infected cells, affecting pathways involved in survival, stress, TGFbeta, p53, NFkappaB, protein kinase C and hedgehog signaling.