Structural and biochemical evaluation of bisubstrate inhibitors of protein arginine N-methyltransferases PRMT1 and CARM1 (PRMT4)

Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the co-factor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts

Identification of lipases with activity towards monoacylglycerol by criterion of conserved cap architectures

Monoacylglycerol lipases (MGL) are a subclass of lipases that predominantly hydrolyze monoacylglycerol (MG) into glycerol and fatty acid. MGLs are ubiquitous enzymes across species and play a role in lipid metabolism, affecting energy homeostasis and signaling processes. Structurally, MGLs belong to the α/β hydrolase fold family with a cap covering the substrate binding pocket. Analysis of the known 3D structures of human, yeast and bacterial MGLs revealed striking similarity of the cap architecture. Since MGLs from different organisms share very low sequence similarity, it is difficult to identify MGLs based on the amino acid sequence alone. Here, we investigated whether the cap architecture could be a characteristic feature of this subclass of lipases with activity towards MG and whether it is possible to identify MGLs based on the cap shape. Through database searches, we identified the structures of five different candidate α/β hydrolase fold proteins with unknown or reported esterase activity. These proteins exhibit cap architecture similarities to known human, yeast and bacterial MGL structures. Out of these candidates we confirmed MGL activity for the protein LipS, which displayed the highest structural similarity to known MGLs. Two further enzymes, Avi_0199 and VC1974, displayed low level MGL activities. These findings corroborate our hypothesis that this conserved cap architecture can be used as criterion to identify lipases with activity towards MGs

Identification of lipases with activity towards monoacylglycerol by criterion of conserved cap architectures

Monoacylglycerol lipases (MGL) are a subclass of lipases that predominantly hydrolyze monoacylglycerol (MG) into glycerol and fatty acid. MGLs are ubiquitous enzymes across species and play a role in lipid metabolism, affecting energy homeostasis and signaling processes. Structurally, MGLs belong to the α/β hydrolase fold family with a cap covering the substrate binding pocket. Analysis of the known 3D structures of human, yeast and bacterial MGLs revealed striking similarity of the cap architecture. Since MGLs from different organisms share very low sequence similarity, it is difficult to identify MGLs based on the amino acid sequence alone. Here, we investigated whether the cap architecture could be a characteristic feature of this subclass of lipases with activity towards MG and whether it is possible to identify MGLs based on the cap shape. Through database searches, we identified the structures of five different candidate α/β hydrolase fold proteins with unknown or reported esterase activity. These proteins exhibit cap architecture similarities to known human, yeast and bacterial MGL structures. Out of these candidates we confirmed MGL activity for the protein LipS, which displayed the highest structural similarity to known MGLs. Two further enzymes, Avi_0199 and VC1974, displayed low level MGL activities. These findings corroborate our hypothesis that this conserved cap architecture can be used as criterion to identify lipases with activity towards MGs

Identification of lipases with activity towards monoacylglycerol by criterion of conserved cap architectures

Monoacylglycerol lipases (MGL) are a subclass of lipases that predominantly hydrolyze monoacylglycerol (MG) into glycerol and fatty acid. MGLs are ubiquitous enzymes across species and play a role in lipid metabolism, affecting energy homeostasis and signaling processes. Structurally, MGLs belong to the α/β hydrolase fold family with a cap covering the substrate binding pocket. Analysis of the known 3D structures of human, yeast and bacterial MGLs revealed striking similarity of the cap architecture. Since MGLs from different organisms share very low sequence similarity, it is difficult to identify MGLs based on the amino acid sequence alone. Here, we investigated whether the cap architecture could be a characteristic feature of this subclass of lipases with activity towards MG and whether it is possible to identify MGLs based on the cap shape. Through database searches, we identified the structures of five different candidate α/β hydrolase fold proteins with unknown or reported esterase activity. These proteins exhibit cap architecture similarities to known human, yeast and bacterial MGL structures. Out of these candidates we confirmed MGL activity for the protein LipS, which displayed the highest structural similarity to known MGLs. Two further enzymes, Avi_0199 and VC1974, displayed low level MGL activities. These findings corroborate our hypothesis that this conserved cap architecture can be used as criterion to identify lipases with activity towards MGs

Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase

Lactate dehydrogenase A (LDH-A) is a key enzyme in anaerobic respiration that is predominantly found in skeletal muscle and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH. LDH-A is overexpressed in many tumours and has therefore emerged as an attractive target for anticancer drug discovery. Crystal structures of human LDH-A in the presence of inhibitors have been described, but currently no structures of the apo or binary NADH-bound forms are available for any mammalian LDH-A. Here, the apo structure of human LDH-A was solved at a resolution of 2.1 Å in space group P4122. The active-site loop adopts an open conformation and the packing and crystallization conditions suggest that the crystal form is suitable for soaking experiments. The soaking potential was assessed with the cofactor NADH, which yielded a ligand-bound crystal structure in the absence of any inhibitors. The structures show that NADH binding induces small conformational changes in the active-site loop and an adjacent helix. A comparison with other eukaryotic apo LDH structures reveals the conservation of intra-loop interactions. The structures provide novel insight into cofactor binding and provide the foundation for soaking experiments with fragments and inhibitors

Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets

Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening

Plasma kallikrein structure reveals apple domain disc rotated conformation compared to factor XI

BackgroundPlasma prekallikrein (PK) and factor XI (FXI) are apple domain‐containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways.ObjectiveTo investigate the three‐dimensional structure of full‐length PKa and perform a comparison with FXI.MethodsA series of recombinant full‐length PKa and FXI constructs and variants were developed and the crystal structures determined.Results and conclusionsA 1.3 Å structure of full‐length PKa reveals the protease domain positioned above a disc‐shaped assemblage of four apple domains in an active conformation. A comparison with the homologous FXI structure reveals the intramolecular disulfide and structural differences in the apple 4 domain that prevents dimer formation in PK as opposed to FXI. Two latchlike loops (LL1 and LL2) extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen‐deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation

Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery

The contact system is composed of Factor XII (FXII), prekallikrein (PK) and co-factor kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+ dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion with residues Arg36 and Arg65 forming contacts with two distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII binding site and a comparison with the ligand free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+ binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with SPR demonstrate the gC1qR Zn2+ site contributes to FXII binding and plasma based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only one high affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher order 500kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors which may be a prelude for initiating the cascades which drive bradykinin generation and the intrinsic pathway of coagulation

Structures of factor XI and prekallikrein bound to domain 6 of high–molecular weight kininogen reveal alternate domain 6 conformations and exosites

High molecular weight kininogen (HK) circulates in plasma as a complex with zymogen prekallikrein (PK). HK is both substrate and co-factor for activated plasma kallikrein (PKa) and the principal exosite interactions occur between PK N-terminal apple domains and the C-terminal D6 domain of HK. To determine the structure of the complex formed between PK apple domains and a HKD6 fragment and compare this to the FXI-HK complex. We produced recombinant FXI and PK heavy chains (HC) spanning all four apple domains. We co-crystallised PKHC (and subsequently FXIHC) with a 31 amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analysed the full length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry (HDX-MS). The 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3. The alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI. [Abstract copyright: Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel

© 2018 Ward et al. Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail-binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1' region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1' binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development