Kellner Julian N, Reinstein Jochen, Meinhart Anton
Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany
Nucleic Acids Res. 2015 Mar 11;43(5):2813-28. doi: 10.1093/nar/gkv106. Epub 2015 Feb 17.
RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1's enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an 'open' to a 'closed'-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.
DEAD-box蛋白家族的RNA解旋酶构成了解旋酶的最大群体。人类DEAD-box蛋白1（DDX1）在tRNA和mRNA加工过程中发挥重要作用，参与肿瘤进展，还被包括HIV-1在内的多个病毒家族劫持用于复制和核输出。尽管DDX1在许多细胞过程中都很重要，但其酶促功能的机制尚不清楚。我们进行了平衡滴定和瞬态动力学实验，以确定其对核苷酸和RNA的亲和力。我们发现DDX1与二磷酸腺苷（ADP）具有异常紧密的结合，这是观察到的DEAD-box解旋酶最强的亲和力之一。与三磷酸腺苷（ATP）相比，ADP的结合力强三个数量级，在生理条件下使酶处于潜在的ADP终产物构象。因此，我们认为核苷酸交换因子会导致DDX1的循环利用。此外，我们发现RNA和ATP与DDX1的结合具有很强的协同性，这也反映在ATP水解过程中。我们提出了一个模型，其中单独的ATP或RNA结合都可以部分地将平衡从“开放”状态转变为“封闭”状态；即使在同时存在RNA和ATP的情况下，这种转变似乎也不会进一步显著，因为ATP水解的低速率没有变化。
