Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

Receptor-interacting serine/threonine-protein kinase 1(RIPK1)functions as a key regulator in inflammation and cell death and is involved in mediating a variety of inflammatory or degenerative diseases.A number of allosteric RIPK1 inhibitors(RIPK1i)have been developed,and some of them have already advanced into clinical evaluation.Recently,selective RIPK1i that interact with both the allosteric pocket and the ATP-binding site of RIPK1 have started to emerge.Here,we report the rational development of a new series of type-II RIPK1i based on the rediscovery of a reported but mechanistically atypical RIPK3i.We also describe the structure-guided lead optimization of a potent,selective,and orally bioavailable RIPK1i,62,which exhibits extraordinary efficacies in mouse models of acute or chronic inflammatory diseases.Collectively,62 provides a useful tool for evaluating RIPK1 in animal disease models and a promising lead for further drug development.

Mechanistic Investigations into the Catalytic Mode of a Dehydratase Complex Involved in the Biosynthesis of Lantibiotic Cacaoidin

Dehydration of serine/threonine residues necessitates the activity of a dehydratase enzyme(domain)during the biosynthesis of RiPP.Recently,it was reported that dehydration in the thioviridamide pathway relies on a distinct dehydratase complex that showcases the activities of a phosphotransferase TvaC for serine/threonine phosphorylation and a lyase TvaD for subsequent phosphate elimination.Herein,we report that dehydration reactions in the pathway of lantibiotic cacaoidin involves a similar dehydratase complex,CaoK/CaoY.Remarkably,this dehydratase complex exhibits flexible enzymatic activity and tolerates significant variations in its substrate peptide sequence.By binding with the leader peptide(LP)sequence of precursor peptide CaoA,the dehydration reactions proceed in a directional manner from the C-terminus of the core peptide(CP)to the N-terminus,and C-terminally truncated variants of CP are acceptable.We show that fusing CaoK to CaoY in a 1:1 molar ratio enables the resulting enzyme CaoYK to exert enhanced dehydration activity.CaoK binds with the LP to improve its own solubility and to ensure the phosphate transfer activity,while CaoY functions in a manner independently of LP.This work advances our understanding of the dehydration process during cacaoidin formation,and provides useful enzymes and methods for the studies of the rapidly emerging RiPPs.

Extensions of PDZ domains as important structural and functional elements

‘Divide and conquer’has been the guiding strategy for the study of protein structure and function.Proteins are divided into domains with each domain having a canonical structural definition depending on its type.In this review,we push forward with the interesting observation that many domains have regions outside of their canonical definition that affect their structure and function;we call these regions‘extensions’.We focus on the highly abundant PDZ(PSD-95,DLG1 and ZO-1)domain.Using bioinformatics,we find that many PDZ domains have potential extensions and we developed an openly-accessible website to display our results(http://bcz102.ust.hk/pdzex/).We propose,using well-studied PDZ domains as illustrative examples,that the roles of PDZ extensions can be classified into at least four categories:1)protein dynamics-based modulation of target binding affinity,2)provision of binding sites for macro-molecular assembly,3)structural integration of multi-domain modules,and 4)expansion of the target ligand-binding pocket.Our review highlights the potential structural and functional importance of domain extensions,highlighting the significance of looking beyond the canonical boundaries of protein domains in general.

Mechanistic lnsights into the Interactions of Ras Subfamily GTPases withthe SPN Domain of Autism-associated SHANK3

of main observation and conclusion The active Ras subfamily GTPases,R apl and Ras,can be specifically recognized by the SPN domain of SHANK3,mutations of which are associated with many neuropsychiatric diseases such as autism spectrum disorder(ASD).However,the mechanistic bases underlying the interactions of SHANK3 SPN and those Ras subfamily proteins are still elusive.Here,we reported the crystal structures of SHANK3 SPN in complex with the GTP-bound Raplb and the Ras-mimetic Raplb E30D/K31E double mutant.In addition to uncovering the detailed molecular mechanism governing the specific interactions of SHANK3 SPN with those Ras subfamily proteins,the determined structures also reveal a general binding mode between SHANK3 SPN and its associated Ras subfamily proteins.Finally,our study also provides mechanistic insights into two ASD-causing R12C and L68P mutations found in the SPN domain of SHANK3,and expands our understanding of the etiology of neuropsychiatric diseases caused by defective SHANK3.