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  • p-Cresyl sulfate br Materials and methods br Results br Disc

    2021-04-30


    Materials and methods
    Results
    Discussion The studies reported here provide a more direct link between conformations of the glycine-rich region of DAPK and enzyme activity. As schematically illustrated in Fig. 3, the various conformations of DAPK with bound substrate or product can be considered reflective of transient states in a catalytic cycle. The protein kinase binds the two substrates, ATP and protein, and facilitates a phosphoryl transfer reaction, followed by release of products. The phosphoryl transfer step is fast, while the release of products is the rate-limiting step [18]. The demonstration that a point mutation in the glycine-rich region can result in only localized changes in structure yet diminish catalytic efficiency significantly is consistent with the p-Cresyl sulfate that this loop is a critical modulator of DAPK catalytic activity. As a logical extension of the results presented here, we also explored the potential impact of a Q23K mutation corresponding to the closely related MLCK region. This point mutation was also detrimental to catalytic activity due mainly to the kcat term (data not shown) and also resulted in only localized conformational differences (see PDB IDs: 3DFC, 3DGK). Superimposition of DAPK-AMPPNP (magenta), DAPKQ23V-AMPPNP (grey), and DAPKQ23K-AMPPNP (cyan) reveal (Fig. 4) a common trend of a more closed state for the glycine-rich loop in the mutants, and altered geometry of the nucleotide at the γ-phosphate. Clearly, the glycine-rich loop region around Q23 plays an important role in catalysis, but the manner in which this particular residue contributes to alteration of loop conformation is not as evident. Results from examination of this corresponding region in other Ser/Thr protein kinases are consistent with those reported here. For example, mutagenesis of Ser53 in cAMP-dependent protein kinase (PKA), corresponding to Q23 of DAPK, resulted in a loss of kinase activity [19]. Similarly, PhosK, exhibits a loss of activity when V29, corresponding to Q23, is mutated to a serine [20]. The PhosK-V29S mutant had an apparent increase in affinity for ADP. It was suggested [20] that the apparent increase in affinity was due to an altered conformation leading to binding of the nucleotide product with greater affinity. Our kinetic and structural results for DAPK Q23 mutant are consistent with such models. The hypothesis derived from the work of McNamara et al. [1] was that the portion of the glycine-rich region around Q23 may be essential for interaction with ATP and ADP, making it important in catalytic activity. The current data presented for the DAPKQ23V mutant support this hypothesis. A logical extension of the hypothesis based on the data presented here is that conformational selection around the glycine-rich region of DAPK might be a potential mechanism for modulating kinase activity. For example, restricting conformational sampling by this region of DAPK could result in restriction of phosphate transfer or generation of a more closed conformation. Mechanistically, the latter could result in a decreased release of product, whereas the former could derive from the inability of the Q23 side chain to orient and stabilize the ligand or from an alteration of the geometry around the phosphates such that a less than ideal arrangement would be provided for the phosphoryl transfer.
    Conclusions
    Acknowledgements
    Introduction Inflammatory gene expression is subject to control by an array of stimuli using diverse molecular mechanisms. Transcriptional regulation of macrophage inflammatory gene expression by cytokines is well established, but much recent attention has focused on posttranscriptional mechanisms (Lindemann et al., 2005). In most cases, posttranscriptional regulation reduces gene expression, acting as a counteracting mechanism that limits the inflammatory response or resolves it after clearance of the initiating stimulus (Kracht and Saklatvala, 2002). Translational control mechanisms offer precise regulation of gene expression, economical use of resources (i.e., degradation of protein or mRNA is not required), and the possibility of rapid reversibility (Gebauer and Hentze, 2004, Mazumder et al., 2003b, Sonenberg and Hinnebusch, 2007). Global translational control regulates a majority of genes in response to extracellular stimuli, whereas transcript-selective translational control regulates expression of a specific gene subset. Transcript-selective translational control is generally mediated by the binding of a protein, protein complex, or microRNA to a defined structural element in either the 5′- or 3′UTR of target mRNAs. Similar sequences and structural elements in multiple transcripts are recognized by the same RNA-binding protein(s) to enable coregulation of translation, thereby constituting a posttranscriptional regulon (Keene, 2007, Lindemann et al., 2005).