Unconventional myosins serve in intracellular movements

cNAc-b4-Man trisaccharide. Loss of DrPOMK in Danio Rerio also leads to disrupted muscle function. We crystallized DrPOMK kinase domain and determined its structure in complex with Mg/ADP, aluminum fluoride, and GalNAc-b3 GlcNAc-b4-Man at 2.0 A resolution. Seven conserved Cys are present in POMK homologues, and six of them are involved in forming three pairs of disulfide bridges in DrPOMK. Cys53Cys66 is located in a long loop in the backside of the N-lobe. Cys72-Cys139 connects helix aB and strand b4. Cys201-Cys241 links the catalytic loop with the activation segment. Cys310 alone exists as a free cysteine and is buried in the C-lobe, not exposed to the solvent. DrPOMK kinase has a bilobal architecture characteristic of EPKs, and can be superimposed onto PKA with a rmsd of 3.1 A over 222 aligned Ca atoms. The N-lobe of DrPOMK highly resembles that of PKA, containing a five-stranded b-sheet coupled to the aC helix. The C-lobe is more divergent. Although Salianic acid A web strands b7-b8 and helices aE-aF closely correspond to the equivalent structure elements in PKA, helices aD, aH, and aI of DrPOMK exhibit significant differences in conformation and length. Helix aG is absent in DrPOMK, whereas an a-helix is uniquely present in its catalytic loop. Active site structure In POMK, a Ser occupies the position of the critical Lys72PKA in strand b3. The critical role of this Lys in DrPOMK is instead served by Lys91DrPOMK located at the beginning of strand b2 that reaches into the active site and interacts with the phosphate groups of ADP. Mutation of the corresponding Lys in HsPOMK to Gly completely abolished kinase activity. The catalytic activity is restored in a double mutant, K93G/S108K, which reinstalls the Lys in strand b3. In fact, this mutant, having both the Gly-rich loop and the b3 Lys restored to `normal’, has enhanced kinase activity in vitro compared to the wild-type enzyme. Another important Lys involved in nucleotide-binding is Lys208DrPOMK located in helix aCL. Similar to Lys168PKA, it interacts with the AlF3 group that mimics the transition state g-phosphate of ATP. Mutation of the equivalent Lys in HsPOMK to Ala also significantly impairs catalysis. In most kinases, Glu91PKA forms an ion pair with Lys72PKA, which is important for an active kinase. In POMK, Glu91PKA in the aC helix is replaced by a Gly. Lys91DrPOMK forms an ion pair with Asp227DrPOMK, which occupies the position of the DFG Gly. However, mutation of the homologous Asp to Gly in HsPOMK has no negative effect on catalysis. Furthermore, restoration of the DFG Gly and the aC Glu eliminated kinase activity. Thus, unlike in canonical kinases, an ion pair appears not to be required for the activity of POMK. The catalytic loop and the activation segment, separated by a pair of anti-parallel b-strands, are located in the linker region between helices aE and aF. Compared to PKA, the linkers between helices aE and aF are much longer in POMK, and the catalytic Asp is located in a MCD motif in POMK. Mutation of this Asp in HsPOMK to Ala eradicated kinase activity, corroborating its critical catalytic function. Like the His/Tyr in the Zhu et al. eLife 2016;5:e22238. DOI: 10.7554/eLife.22238 3 of 18 Research article Biochemistry Biophysics and Structural Biology H/YRD motif, the Met in the MCD is involved in forming the regulatory spine structure that stabilizes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826115 the active kinase conformation . In kinases that undergo phosphorylation-dependent activation such as PKA, the H/YRD Arg interacts with

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