Aids Related Nmr Research

这个项目利用核磁共振光谱学来研究HIV和模型系统的分子组成。最近的研究集中在：1)分析HIV逆转录酶核糖核酸酶H(RNaseH)结构域的溶液构象和动力学，以及底物诱导活性部位形成的特征；2)了解蛋白质介导的RT底物和抑制物的相互作用；3)分析从p66和其他早期形式的多聚体到形成成熟的异二聚体结构的构象过程。 HIV-1逆转录酶(RT)是HIV生命周期中的一个关键酶，也是一个重要的药物靶标，它经历了复杂的和大部分未知的构象重排，这是其不对称折叠、二聚化和亚基选择性核糖核酸酶H区(RH)蛋白分解的基础。在过去的一年里，我们首次报道了一系列研究中的第一次，这些研究结合了核磁共振光谱、小角X射线散射和X射线结晶学来表征p51和p66单体以及p66/p66‘同源二聚体的构象成熟。P66单体以松散结构的分子形式存在，其中手指/手掌/连接、拇指和RH亚结构通过柔性(无序)连接段连接在一起。最初观察到的同源二聚体是不对称的，包括两个完全折叠的RH结构域，同时表现出与RT异源二聚体类似的其他构象特征。P66‘亚基的RH’结构域经历了选择性的展开，时间常数约为6.5小时，这与由于残基转移到p66‘亚基上的聚合酶’结构域而导致的不稳定一致。随机线圈位置附近共振强度的同时增加具有相似的时间常数。与残基转移假说一致，缺乏两个N-末端残基的分离的RH结构域的结构表现出稳定性降低。这些结果表明，RH‘的展开与同源二聚体的形成是偶联的。 项目2.虽然p66单体的结构和对p66/p66‘同源二聚体进行的核磁共振研究提供了对同源二聚体中发生的早期构象重排的洞察，但由于核磁共振实验的低灵敏度，无法直接观察这些过程。为了填补这一空白，并与计算核心的Lalith Perera博士合作，我们对各种RT结构域进行了分子动力学模拟，以便更好地了解单体中可能发生的短期构象重排。这些研究与所开发的模型的各个方面是一致的，在该模型中，p66单体发生一系列单分子重排，从而形成更开放的构象，然后这种开放的构象能够与作为构象选择过程的结果而存在的其他单体二聚化。一些模拟针对的是手指/手掌的子域。在单体中，用手指中的螺旋A和手掌中的螺旋F定义的这两个亚区之间的角度相当尖锐，45。在解离连接结构域之后，模拟表明，在模拟的最初几百纳秒内，该角度可以增加到70-100范围内的值，这是p66/p51异二聚体中p66亚单位的特征。因此，这些计算支持单分子构象重排的作用，这与我们的构象成熟模型一致。 根据我们最近的研究，p66单体能够形成构象不对称的p66/p66‘同源二聚体，其中包含两个折叠的RH结构域。由于残基从RH‘转移到p66’上的聚合酶‘结构域，p66’上的RH‘结构域容易选择性失稳。我们通过表达RH结构域的结构来模拟这种不稳定性，其中两个N-末端残基不存在，第三个亮氨酸残基突变为蛋氨酸，RH&8710；NT。最近的研究表明，这种结构比完整的RT结构域的稳定性要差得多，这与基于模型的残基转移失稳是一致的。在过去的一年里，我们对RH和截断结构RH&8710；NT进行了氢/氢交换研究。这些研究与N-末端残基对结构域稳定的重要性是一致的，更具体地说，表明在截短结构中，Phe440-Tyr441裂解位点的酰胺交换率大大增加。

This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the solution conformation and dynamics of the ribonuclease H (RNase H) domain of HIV reverse transcriptase, and the characterization of substrate-induced active site formation; 2) understanding the protein-mediated interactions of RT substrates and inhibitors; 3) analysis of the conformational processes involved in going from p66 and other early forms of the polyprotein to formation of the mature heterodimer structure. Project 1. HIV-1 reverse transcriptase (RT), a critical enzyme of the HIV life cycle and an important drug target, undergoes complex and largely uncharacterized conformational rearrangements that underlie its asymmetric folding, dimerization and subunit-selective ribonuclease H domain (RH) proteolysis. During the past year, we reported the first in a series of studies that utilized a combination of NMR spectroscopy, small angle X-ray scattering and X-ray crystallography to characterize the p51 and p66 monomers and the conformational maturation of the p66/p66' homodimer. The p66 monomer exists as a loosely structured molecule in which the fingers/ palm/connection, thumb and RH substructures are connected by flexible (disordered) linking segments. The initially observed homodimer is asymmetric and includes two fully folded RH domains, while exhibiting other conformational features similar to that of the RT heterodimer. The RH' domain of the p66' subunit undergoes selective unfolding with time constant about 6.5 hours, consistent with destabilization due to residue transfer to the polymerase' domain on the p66' subunit. A simultaneous increase in the intensity of resonances near the random coil positions is characterized by a similar time constant. Consistent with the residue transfer hypothesis, a construct of the isolated RH domain lacking the two N-terminal residues is shown to exhibit reduced stability. These results demonstrate that RH' unfolding is coupled to homodimer formation. Project 2. Although the structure of the p66 monomer and the NMR studies performed on the p66/p66' homodimer provide insight into the early conformational rearrangements that occur in the homodimer, the low sensitivity of the NMR experiment precludes the direct observation of these processes. In order to fill this gap, and in collaboration with Dr. Lalith Perera of the computational core, we have performed molecular dynamics simulations on various RT domains in order to better understand the short term conformational rearrangements that can occur in the monomer. These studies are consistent with the various aspects of the model developed, in which a series of unimolecular rearrangements of the p66 monomer occurs which results in formation of a more open conformation, and this open conformation is then able to dimerize with other monomers that are present as a result of a conformational selection process. Some of the simulations targeted the fingers/palm subdomains. In the monomer, the angle between these two subdomains, defined using helices A in the fingers and F in the palm, is rather sharp, 45. After dissociation of the connection domain, simulations show that this angle can increase during the first few hundred nanoseconds of the simulation to values in the 70-100 range that are characteristic of the p66 subunit in the p66/p51 heterodimer. These calculations thus support the role of a unimolecular conformational rearrangement that is consistent with our conformational maturation model. Project 3. According to our recent studies, p66 monomers are able to form conformationally asymmetric p66/p66' homodimers that contains two, folded RH domains. The RH' domain on p66' is the subject to selective destabilization as a result of the transfer of residues from RH' to the polymerase' domain on p66'. We have modeled this destabilization by expressing a construct of the RH domain in which the two N-terminal residues are not present, and the third leucine residue is mutated to a methionine, RH∆NT. Recent studies demonstrate that this construct is considerably less stable than the full RT domain, consistent with the model-based destabilization by residue transfer. During the past year, we performed hydrogen/deuterium exchange studies on both RH and the truncated construct, RH∆NT. These studies are consistent with the importance of the N-terminal residues for domain stabilization, and more specifically, show that the amide exchange rate at the Phe440-Tyr441 cleavage site is greatly increased in the truncated construct.