The p66 immature precursor of HIV-1 reverse transcriptase. our data provide unique insights into the conformational changes in p66/p66 that drive PR cleavage. An eTOC blurb Slack et al. characterize conformational changes involved in the maturation of HIV-1 reverse transcriptase using NMR spectroscopy. Biochemical and virological experiments are carried out to explain how these factors affect the maturation. Graphical Abstract INTRODUCTION Efficient maturation of HIV-1 proteins is critical for computer virus replication. HIV-1 reverse transcriptase (RT) is usually expressed as part of the viral Gag-Pol polyprotein, which is usually cleaved by HIV-1 protease (PR) to finally form a mature RT heterodimer composed of 66 (p66) and 51 kDa (p51) subunits (p66/p51) (Physique 1A) (Coffin et al., 1997; Katz and Skalka, 1994). The p51 subunit is usually generated upon removal of most of the ribonuclease H (RNH) domain name from p66 (Chattopadhyay et al., 1992; Divita et al., 1995; Sharma et al., 1994). Mouse monoclonal antibody to SAFB1. This gene encodes a DNA-binding protein which has high specificity for scaffold or matrixattachment region DNA elements (S/MAR DNA). This protein is thought to be involved inattaching the base of chromatin loops to the nuclear matrix but there is conflicting evidence as towhether this protein is a component of chromatin or a nuclear matrix protein. Scaffoldattachment factors are a specific subset of nuclear matrix proteins (NMP) that specifically bind toS/MAR. The encoded protein is thought to serve as a molecular base to assemble atranscriptosome complex in the vicinity of actively transcribed genes. It is involved in theregulation of heat shock protein 27 transcription, can act as an estrogen receptor co-repressorand is a candidate for breast tumorigenesis. This gene is arranged head-to-head with a similargene whose product has the same functions. Multiple transcript variants encoding differentisoforms have been found for this gene Two models of RT maturation have been proposed: a model, in which the p66 and p51 subunits are cleaved independently from Gag-Pol, and a model, in which PR first cleaves p66 from the polyprotein and, following p66 dimerization, the p66/p51 RT heterodimer is usually formed (Figueiredo et al., 2006; Lindhofer et al., 1995; Mattei et al., 2014; Pettit et al., 2004; Pettit et al., 2005b; Sluis-Cremer et al., 2004; Speck et al., 2000; Wapling et al., 2005; Zheng et al., 2015; Zheng et al., 2014). In regard to these models, prior biochemical data, including ours, exhibited that p66/p66 homodimer formation is absolutely necessary for efficient RT maturation, thus supporting the sequential model (Physique 1C) (Abram and Parniak, 2005; Abram et al., 2010; Sluis-Cremer et al., 2004). Paradoxically, the p66/p66 homodimer adopts a symmetrical conformation in answer in which both RNH domains are folded and the p51-RNH cleavage sites are inaccessible to PR (Sharaf et al., 2014). Interestingly, in all structures of the mature p66/p51 heterodimer, the p51-RNH cleavage site is usually sequestered in a p-sheet within the RNH domain name and is inaccessible to PR (Physique 1B) (Davies et al., 1991; Jacobo-Molina and Arnold, 1991; Jacobo-Molina et al., 1993; Kohlstaedt et al., 1992). Consequently, the pathways involved in p66/p51 RT maturation have not been defined. However, characteristic differences between the immature p66/p66 homodimer and the mature p66/p51 heterodimer, such as a ~ 10-fold decrease in the dimer dissociation constant (Sharaf et al., 2014; Sluis-Cremer et al., 2000; Venezia et al., 2006), have led to the hypothesis that significant structural differences exist between these RT proteins. Open in a separate window Physique 1. Structure of p66/p51 HIV-1 RT.(A) Overall structure of the p66/p51 heterodimer. The fingers-palm, thumb, connection, and RNH domains in the p66 subunit are purple, green, yellow, and orange, Flutamide respectively. The p51 subunit is usually white. (B) Structure of the RNH domain name highlighting that this p51-RNH cleavage site (F440-Y441, yellow ribbon) is usually sequestered in the protein core. The RNH active site residues are shown by red sticks. (C) Schematic highlighting how p66/p51 is usually generated from p66/p66 by HIV-1 PR-mediated cleavage. In panels (A) and (B), graphics were generated using the structure of PDB 3MEE (Lansdon et al., 2010); the location of RPV is usually shown by red spheres in (A); locations of the Ile-1 methyl groups that were uniquely observed in the NMR data are shown by pink spheres. These are residues 202 in the fingers-palm domain name, 254 and 259 in the thumb domain name, 393 in the connection domain name, and 434, 495, and 559 in the RNH domain name. Note, since crystallographic coordinates are not available for residue 559, the position of residue 559 is usually approximated. Recently, we developed an RT maturation assay that evaluates.HIV-1 PR, clone purchased from ATUM (Newark, CA), was expressed and purified as described previously (Khan et al., 2018). inhibitors, to modulate the p66 dimerCmonomer equilibrium and monitor the resulting structural changes. Taken together, our data provide unique insights into the conformational changes in p66/p66 that drive PR cleavage. An eTOC blurb Slack et al. characterize conformational changes involved in the maturation of HIV-1 reverse transcriptase using NMR spectroscopy. Biochemical and virological experiments are carried out to explain how these factors affect the maturation. Graphical Abstract INTRODUCTION Efficient maturation of HIV-1 proteins is critical for computer virus replication. HIV-1 reverse transcriptase (RT) is usually expressed as part of the viral Gag-Pol polyprotein, which is usually cleaved by HIV-1 protease (PR) to finally form a mature RT heterodimer composed of 66 (p66) and 51 kDa (p51) subunits (p66/p51) (Physique 1A) (Coffin et al., 1997; Katz and Skalka, 1994). The p51 subunit is usually generated upon removal of most of the ribonuclease H (RNH) domain name from p66 (Chattopadhyay et al., 1992; Divita et al., 1995; Sharma et al., 1994). Two models of RT maturation have been proposed: a model, in which the p66 and p51 subunits are cleaved independently from Gag-Pol, and a model, in which PR first cleaves p66 from the polyprotein and, following p66 dimerization, the p66/p51 RT heterodimer is formed (Figueiredo et al., 2006; Lindhofer et al., 1995; Mattei et al., 2014; Pettit et al., 2004; Pettit et al., 2005b; Sluis-Cremer et al., 2004; Speck et al., 2000; Wapling et al., 2005; Zheng et al., 2015; Zheng et al., 2014). In regard to these models, prior biochemical data, including ours, demonstrated that p66/p66 homodimer formation is absolutely necessary for efficient RT maturation, thus supporting the sequential model (Figure 1C) (Abram and Parniak, 2005; Abram et al., 2010; Sluis-Cremer et al., 2004). Paradoxically, the p66/p66 homodimer adopts a symmetrical conformation in solution in which both RNH domains are folded and the p51-RNH cleavage sites are inaccessible to PR (Sharaf et al., 2014). Interestingly, in all structures of the mature p66/p51 heterodimer, the p51-RNH cleavage site is sequestered in a p-sheet within the RNH domain and is inaccessible to PR (Figure 1B) (Davies et al., 1991; Jacobo-Molina and Arnold, 1991; Jacobo-Molina et al., 1993; Kohlstaedt et al., 1992). Consequently, the pathways involved in p66/p51 RT maturation have not been defined. However, characteristic differences between the immature p66/p66 homodimer and the mature p66/p51 heterodimer, such as a ~ 10-fold decrease in the dimer dissociation constant (Sharaf et al., 2014; Sluis-Cremer et al., 2000; Venezia et al., 2006), have led to the hypothesis that significant structural differences exist between these RT proteins. Open in a separate window Figure 1. Structure of p66/p51 HIV-1 RT.(A) Overall structure of the p66/p51 heterodimer. The fingers-palm, thumb, connection, and RNH domains in the p66 subunit are purple, green, yellow, and orange, respectively. Flutamide The p51 subunit is white. (B) Structure of the RNH domain highlighting that the p51-RNH cleavage site (F440-Y441, yellow ribbon) is sequestered in the protein core. The RNH active site residues are shown by red sticks. (C) Schematic highlighting how p66/p51 is generated from p66/p66 by HIV-1 PR-mediated cleavage. In panels (A) and (B), graphics were generated using the structure of PDB 3MEE (Lansdon et al., 2010); the location of RPV is shown by red spheres in (A); locations of the Ile-1 methyl groups that were uniquely observed in the NMR data are shown by pink spheres. These are residues 202 in the fingers-palm domain, 254 and 259 in Flutamide the thumb domain, 393 in the connection domain, and 434, 495, and 559 in the RNH domain. Note, since crystallographic coordinates are not available for residue 559, the position of residue 559 is approximated. Recently, we developed an RT maturation assay that evaluates processing of p66 by active HIV-1 PR to.