The structure of the large regulatory α subunit of phosphorylase kinase examined by modeling and hydrogen‐deuterium exchange

Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)4, and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK’s four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full‐length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen‐deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N‐terminal glycoside hydrolase domain and a large C‐terminal importin α/β‐like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen‐deuterium exchange results obtained for this subunit as part of the (αβγδ)4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter‐subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low‐exchanging region in the C‐terminus of α that is known to bind the regulatory domain of the catalytic γ subunit.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141441/1/pro3339.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141441/2/pro3339-sup-0001-suppinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141441/3/pro3339_am.pd

An evaluation of indirubin analogues as phosphorylase kinase inhibitors

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus S. et al., Oncogene 2012, 31, 4333). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170 – 0.360 µM, with indirubin-3’-acetoxime (1c) the most potent. 7-bromoindirubin-3’-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas J. et al., Oncogene, 2006, 25, 6304) is revealed as a specific inhibitor of PhK (IC50 = 1.8 µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting -hole effects is highlighted. A new statistical metric, the ‘sum of the modified logarithm of ranks’ (SMLR), has been defined which measures performance of a model for both the “early recognition” (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6’(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work

Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members