Marti Daniel N, Bjelić Sasa, Lu Min, Bosshard Hans Rudolf, Jelesarov Ilian
Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
J Mol Biol. 2004 Feb 6;336(1):1-8. doi: 10.1016/j.jmb.2003.11.058.
Human (HIV-1) and simian (SIV) immunodeficiency virus fusion with the host cell is promoted by the receptor-triggered refolding of the gp41 envelope protein into a stable trimer-of-hairpins structure that brings viral and cellular membranes into close proximity. The core of this hairpin structure is a six-helix bundle in which an inner homotrimeric coiled coil is buttressed by three antiparallel outer HR2 helices. We have used stopped-flow circular dichroism spectroscopy to characterize the unfolding and refolding kinetics of the six-helix bundle using the HIV-1 and SIV N34(L6)C28 polypeptides. In each case, the time-course of ellipticity changes in refolding experiments is well described by a simple two-state model involving the native trimer and the unfolded monomers. The unfolding free energy of the HIV-1 and SIV trimers and their urea dependence calculated from kinetic data are in very good agreement with data measured directly by isothermal unfolding experiments. Thus, formation of the gp41 six-helix bundle structure involves no detectable population of stable, partly folded intermediates. Folding of HIV-1 N34(L6)C28 is five orders of magnitudes faster than folding of its SIV counterpart in aqueous buffer: k(on),(HIV-1)=1.3 x 10(15)M(-2)s(-1) versus k(on),(SIV)=1.1 x 10(10)M(-2)s(-1). The unfolding rates are similar: k(off),(HIV-1)=1.1 x 10(-5)s(-1) versus k(off),(SIV=)5.7 x 10(-4)s(-1). Kinetic m-values indicate that the transition state for folding of the HIV-1 protein is significantly more compact than the transition state of the SIV protein. Replacement of a single SIV threonine by isoleucine corresponding to position 573 in the HIV-1 sequence significantly stabilizes the protein and renders the folding rate close to that of the HIV-1 protein yet without making the transition state of the mutant as compact as that of the HIV-1 protein. Therefore, the overall reduction of surface exposure in the high-energy transition state seems not to account for different folding rates. While the available biological evidence suggests that refolding of the gp41 protein is slow, our study implies that structural elements outside the trimer-of-hairpins limit the rate of HIV-1 fusion kinetics.
人类免疫缺陷病毒1型（HIV-1）和猿猴免疫缺陷病毒（SIV）与宿主细胞的融合，是由受体触发的gp41包膜蛋白重折叠成稳定的发夹三聚体结构所促进的，该结构使病毒膜和细胞膜紧密靠近。这种发夹结构的核心是一个六螺旋束，其中一个内部同三聚体卷曲螺旋由三个反平行的外部HR2螺旋支撑。我们使用停流圆二色光谱法，以HIV-1和SIV的N34(L6)C28多肽为材料，来表征六螺旋束的解折叠和重折叠动力学。在每种情况下，重折叠实验中椭圆率变化的时间进程，都可以用一个简单的两态模型很好地描述，该模型涉及天然三聚体和未折叠的单体。从动力学数据计算得到的HIV-1和SIV三聚体的解折叠自由能及其对尿素的依赖性，与通过等温解折叠实验直接测量的数据非常吻合。因此，gp41六螺旋束结构的形成不涉及可检测到的稳定的部分折叠中间体群体。在水性缓冲液中，HIV-1的N34(L6)C28的折叠速度比其SIV对应物快五个数量级：k(on),(HIV-1)=1.3×10(15)M(-2)s(-1)，而k(on),(SIV)=1.1×10(10)M(-2)s(-1)。解折叠速率相似：k(off),(HIV-1)=1.1×10(-5)s(-1)，而k(off),(SIV)=5.7×10(-4)s(-1)。动力学m值表明，HIV-1蛋白折叠的过渡态比SIV蛋白的过渡态明显更紧密。将SIV中对应于HIV-1序列第573位的苏氨酸替换为异亮氨酸，可显著稳定该蛋白，并使折叠速率接近HIV-1蛋白的折叠速率，但突变体的过渡态不像HIV-1蛋白的过渡态那么紧密。因此，在高能过渡态中表面暴露的总体减少似乎不能解释不同的折叠速率。虽然现有的生物学证据表明gp41蛋白的重折叠很慢，但我们的研究表明，发夹三聚体之外的结构元件限制了HIV-1融合动力学的速率。
