ИСТИНА

Today, the treatment of HIV infection mostly relies on a combination of inhibitors of viral enzymes (HAART) and as a result, drug resistant viral strains are eventually generated. However, a new approach for HIV drug design is being currently developed that is focused on the disruption of functional interactions between viral enzymes and their cellular co-factors. This approach has been proven to be applicable when compounds that specifically disrupt the interaction between HIV-1 integrase (IN) and its cellular co-factor LEDGF inhibited viral replication in cell culture. Recently, a few reports showed that Ku70, which is a part of the DNA-PK complex, prevents HIV-1 IN from degradation potentially by a direct interaction. It is known, that in cells depleted of Ku70 levels of viral replication are significantly lowered. Thus, the inhibition of Ku70-IN interaction might affect viral replication. A detailed structure of Ku70/IN complex would greatly facilitate drug design. Unfortunately, only single domains of IN can be effectively crystallized. In this regard, it is important to estimate the minimal subdomains within IN and Ku70 that are required for complex formation. A full-size N-His6-tagged HIV-1 IN was purified form E. coli as well as its C-terminal domain (IN_220-270 a.a.). At first, two versions of Ku70, Ku70_wt and a protein carrying the first 430 amino acids of Ku70 (Ku70_430), were purified also from E. coli both carrying a GST-tag on their N-termini. Ku70_wt and Ku70_430 both formed stable complexes with IN with Kd approximated at 80 nM as determined by GST and His6 pull downs. Ku70_wt and Ku70_430 undergo proteolysis during expression with formation of one major N-terminal product (1-319 a.a., identified by mass-spectrometry). Interestingly, this proteolytic product when individually purified interacts with IN_wt as strongly as do larger proteins with Kd approximated at 70 nM. This deletion mutant of Ku70 also equally binds the C-terminal domain of IN. The X-ray structure of Ku70 shows that Ku70_430 contains the N-terminal domain and the DNA-binding ring stabilized by a β-barrel formed by N- and C- terminal a.a., while Ku70_319 contains just the N-terminal domain and a part of the DNA-binding loop. To investigate the function of DNA-binding loop in complex formation we constructed and purified additional Ku70 deletion mutants: Ku70_309, Ku70_294, Ku70_273 and Ku70_250, where the later comprised the sole N-terminal domain of Ku70. All of them co-precipitated with IN_wt. Therefore, the DNA-binding ring in Ku70 is not an essential structure for IN/Ku70 complex formation. Also, we successfully minimized the fractions of Ku70 and IN that are required for complex formation.