Bax, B. D. | bioRxiv Science

bioRxiv2021

Structures of the Human SPAK and OSR1 Conserved C-Terminal (CCT) Domains

STE20/SPS1-related proline/alanine-rich kinase (SPAK) and Oxidative Stress Responsive 1 (OSR1) kinase are two serine/threonine protein kinase that regulate the function of ion co-transporters through phosphorylation. The highly conserved C-terminal (CCT) domains of SPAK and OSR1 bind to RFx[V/I] peptide sequences from their upstream With No Lysine Kinases (WNKs), facilitating their activation via phosphorylation. Thus, the inhibition of SPAK and OSR1 binding, via their CCT domains, to WNK kinases is a plausible strategy for inhibiting SPAK and OSR1 kinases. To facilitate structure-guided drug design of such inhibitors, we expressed and purified human SPAK and OSR1 CCT domains and solved their crystal structures. We also employed a biophysical strategy and determined the affinity of SPAK and OSR1 CCT domains to an 18-mer peptide derived from WNK4. Together, the crystal structures and affinity data reported herein provide a robust platform to facilitate the design of CCT domain specific small molecule inhibitors of SPAK-activation by WNK kinases, potentially leading to new improved treatments for hypertension and ischemic stroke.

biochemistry↗

bioRxiv2021

Tyrosine 121 moves revealing a druggable pocket that couples catalysis to ATP-binding in serine racemase

Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a closed hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the ATP a-phosphate. In contrast, in open hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzymefragment structures show that Tyr121 is flipped out of its pocket, suggesting that this pocket is druggable.

biophysics↗