Scherr Nicole, Honnappa Srinivas, Kunz Gabriele, Mueller Philipp, Jayachandran Rajesh, Winkler Fritz, Pieters Jean, Steinmetz Michel O
Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):12151-6. doi: 10.1073/pnas.0702842104. Epub 2007 Jul 6.
The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG with the highly selective low-molecular-weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here, we report the 2.4 A x-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis.
结核分枝杆菌等分枝杆菌的致病性与其在宿主巨噬细胞内存活的能力密切相关。细胞内分枝杆菌存活的一个关键毒力因子是蛋白激酶G（PknG），它是一种由致病性分枝杆菌表达的类似真核生物的丝氨酸/苏氨酸蛋白激酶，可阻止分枝杆菌在溶酶体中的细胞内降解。用高选择性低分子量抑制剂AX20017抑制PknG会导致分枝杆菌转移至溶酶体并被杀死。在此，我们报告了PknG与AX20017复合物的2.4埃X射线晶体结构。PknG独特的多结构域拓扑结构显示，中央激酶结构域两侧分别是N端和C端的铁氧化还原蛋白及四三肽重复结构域。定向诱变表明，铁氧化还原蛋白结构域作为PknG激酶活性的调节剂发挥作用。PknG-AX20017的结构进一步显示，抑制剂深埋于腺苷结合位点内，靶向激酶结构域的活性构象。值得注意的是，尽管激酶结构域的拓扑结构让人联想到真核生物激酶，但AX20017结合口袋是由一组在任何人类激酶中都未发现的独特氨基酸侧链形成的。对这组独特残基进行定向诱变导致该化合物的抑制效力大幅丧失。我们的结果解释了AX20017的具体作用方式，并证明与宿主分子高度同源的毒力因子可以成功作为靶点来阻断结核分枝杆菌的增殖。
