Chen Yueyi, Tesmer John J G
Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA; Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, USA.
Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA; Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, USA.
J Biol Chem. 2025 May 29;301(7):110309. doi: 10.1016/j.jbc.2025.110309.
G protein-coupled receptor (GPCR) kinases (GRKs) instigateGPCR desensitization, but despite many available structures, a molecular understanding of their function and catalytic cycle remains incomplete. We present six GRK5 crystal structures that capture both open and closed states of its kinase domain as well as complexes with the ligands sangivamycin (Sgv), an adenosine analog, and ATP. The Sgv-bound structure is distinct from the previously reported GRK5·Sgv structure and features an ordered N-terminal helix that docks to the kinase hinge, mimicking its interactions in GPCR or Ca·calmodulin-bound GRK complexes. GRK5 undergoes a dramatic conformational change in the crystals to a ligand-free, open state with a disordered N terminus when K is omitted from the harvesting solution. This transition to a ligand-free structure, not structurally observed for the GRK4 subfamily, most likely occurs through the release of the K ion from its binding site close to the kinase domain hinge in the Sgv-bound complex. Two structures of GRK5 in complex with Mg and Mn·ATP were obtained via soaking crystals of the open state, which we hypothesize are reflective of a substrate-loading stage. Although K significantly stabilizes GRK5 in its closed, near-active conformation, potassium citrate and KCl inhibit kinase activity just as potently as sodium citrate and NaCl, respectively, suggesting that K traps a closed conformation compatible with Sgv-AMP but incompatible with ATP, thereby inhibiting the catalytic cycle. Thus, changes in K concentration could play a regulatory role for GRK5 in scenarios where activated GPCRs are coupled to G protein-responsive potassium channels.
G蛋白偶联受体（GPCR）激酶（GRK）引发GPCR脱敏，但尽管有许多可用结构，对其功能和催化循环的分子理解仍不完整。我们展示了六个GRK5晶体结构，这些结构捕获了其激酶结构域的开放和关闭状态以及与配体桑吉瓦霉素（Sgv，一种腺苷类似物）和ATP的复合物。与Sgv结合的结构与先前报道的GRK5·Sgv结构不同，其特征是一个有序的N端螺旋与激酶铰链对接，模拟其在GPCR或钙调蛋白结合的GRK复合物中的相互作用。当从收获溶液中省略钾时，GRK5在晶体中会发生剧烈的构象变化，转变为无配体的开放状态，N端无序。这种向无配体结构的转变在GRK4亚家族中未在结构上观察到，最有可能是通过从其在与Sgv结合的复合物中靠近激酶结构域铰链的结合位点释放钾离子而发生的。通过浸泡开放状态的晶体获得了两个与镁和锰·ATP结合的GRK5结构，我们假设这反映了底物加载阶段。尽管钾能显著稳定GRK5的关闭、近活性构象，但柠檬酸钾和氯化钾分别与柠檬酸钠和氯化钠一样有效地抑制激酶活性，这表明钾捕获了一种与Sgv-AMP兼容但与ATP不兼容的关闭构象，从而抑制了催化循环。因此，在激活的GPCR与G蛋白反应性钾通道偶联的情况下，钾浓度的变化可能对GRK5起调节作用。
