Crystal structures of human ENPP1 in apo and bound forms

Dennis, Matthew L.; Newman, Janet; Dolezal, Olan; Hattarki, Meghan; Surjadi, Regina; Nuttall, Stewart D.; Pham, Tam; Nebl, Thomas; Camerino, Michelle; Khoo, Poh Sim; Monahan, Brendon J.; Peat, T.S.

doi:10.1107/s2059798320010505

Cited by 25 publications

(36 citation statements)

References 35 publications

(35 reference statements)

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“…The bimetallic zinc binding site in ENPP1-7. a The bimetallic zinc binding site as found in PDB-REDO models (PDB identifiers for ENPP1-7: 6weu 27 , 5mhp 28 , 6c01 29 , 4lqy 30 , 5veo 31 , 5egh 32 , 5tcd 33 , respectively) b The same binding site as found in the human ENPP1-7 models from AlphaFold. c The bimetallic zinc binding site in the human ENPP1-7 models as available in AlphaFill, containing the two zinc ions.…”

Section: Resultsmentioning

confidence: 88%

AlphaFill: enriching the AlphaFold models with ligands and co-factors

Hekkelman

Vries

Joosten

et al. 2021

Preprint

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Artificial intelligence (AI) methods for constructing structural models of proteins on the basis of their sequence are having a transformative effect in biomolecular sciences. The AlphaFold protein structure database makes available hundreds of thousands of protein structures. However, all these structures lack cofactors essential for their structural integrity and molecular function (e.g. hemoglobin lacks a bound heme), key ions essential for structural integrity (e.g. zinc-finger motifs) or catalysis (e.g. Ca2+ or Zn2+ in metalloproteases), and ligands that are important for biological function (e.g. kinase structures lack ADP or ATP). Here, we present AlphaFill, an algorithm based on sequence and structure similarity, to “transplant” such “missing” small molecules and ions from experimentally determined structures to predicted protein models. These publicly available structural annotations are mapped to predicted protein models, to help scientists interpret biological function and design experiments.

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Section: Resultsmentioning

confidence: 88%

AlphaFill: enriching the AlphaFold models with ligands and co-factors

Hekkelman

Vries

Joosten

et al. 2021

Preprint

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“…It was particularly surprising that mutating H362 to a variety of amino acids still preserved ATP activity, given that this histidine seems to be chelating one of the two essential zinc atoms in the crystal structures (Dennis et al, 2020; Jansen et al, 2012; Kato et al, 2012, 2018). Additionally, this histidine is highly conserved in members of the NPP and alkaline phosphatase superfamily, which extends from mammals to bacteria (Gijsbers et al, 2001; Zalatan et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Probing pathophysiology of extracellular cGAMP with substrate-selective ENPP1

Cordova

AlSaif

et al. 2021

Preprint

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The biology of the immune second messenger cGAMP depends on its cellular localization. cGAMP, which is synthesized in response to cytosolic double-stranded DNA, also exists in the extracellular space as a paracrine immunotransmitter that enhances the anticancer immune response. However, the role of extracellular cGAMP is unexplored outside of cancer due to a lack of tools to systemically manipulate it. The extracellular enzyme ENPP1, the only known hydrolase of cGAMP, is a promising target. However, because ENPP1 also degrades extracellular ATP, using genetic knockouts of ENPP1 to study extracellular cGAMP leads to confounding effects. Here we report the H362A point mutation in ENPP1, the dominant cGAMP hydrolase, which selectively abolishes ENPP1’s ability to degrade cGAMP, while retaining activity toward other substrates. H362 is not necessary for binding cGAMP or the catalytically-essential zinc atoms but instead supports the in-line reaction geometry. H362 is evolutionarily conserved down to bacteria, suggesting an ancient origin for extracellular cGAMP biology. Enpp1H362A mice do not display the systemic calcification seen in Enpp1-/- mice, highlighting the substrate-specific phenotype of ENPP1. Remarkably, Enpp1H362A mice were resistant to HSV-1 infection, demonstrating the antiviral role of endogenous extracellular cGAMP. The ENPP1H362A mutation is the first genetic tool to enable exploration of extracellular cGAMP biology in a wide range of tissues and diseases.

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“…Different promiscuous enzymes rely on water-bridged ligand interactions for their differential substrate binding, such as RNAses (Ivanov et al, 2019), N-succinyl-amino-acid racemases (Martı ´nez-Rodrı ´guez et al, 2020) and cytochrome P450 (Madrona et al, 2013). This feature has also been observed for promiscuous solute-binding proteins (Clifton & Jackson, 2016;Matsuoka et al, 2015;Camara-Artigas et al, 2016), and in fact other AP superfamily members present water-mediated ligand interactions, such as the promiscuous ectonucleotidase NPP1 (Namasivayam et al, 2017;Dennis et al, 2020), endo-4S--carrageenan sulfatase (Hettle et al, 2018) and N-acetylgalactosamine-6-O-sulfatase (Ndeh et al, 2020). Our structural models clearly support the involvement of water-mediated interactions assisting choline positioning after hydrolysis, where Lys309, Asp386 and Asn75 might assist in leaving-group stabilization (Figs.…”

Section: Mechanistic Implications For the Activity Of Smecosementioning

confidence: 92%

Structural insights into choline-O-sulfatase reveal the molecular determinants for ligand binding

Gavira

Cámara-Artigas

Neira

et al. 2022

Acta Cryst Sect D Struct Biol

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Choline-O-sulfatase (COSe; EC 3.1.6.6) is a member of the alkaline phosphatase (AP) superfamily, and its natural function is to hydrolyze choline-O-sulfate into choline and sulfate. Despite its natural function, the major interest in this enzyme resides in the landmark catalytic/substrate promiscuity of sulfatases, which has led to attention in the biotechnological field due to their potential in protein engineering. In this work, an in-depth structural analysis of wild-type Sinorhizobium (Ensifer) meliloti COSe (SmeCOSe) and its C54S active-site mutant is reported. The binding mode of this AP superfamily member to both products of the reaction (sulfate and choline) and to a substrate-like compound are shown for the first time. The structures further confirm the importance of the C-terminal extension of the enzyme in becoming part of the active site and participating in enzyme activity through dynamic intra-subunit and inter-subunit hydrogen bonds (Asn146 A –Asp500 B –Asn498 B ). These residues act as the `gatekeeper' responsible for the open/closed conformations of the enzyme, in addition to assisting in ligand binding through the rearrangement of Leu499 (with a movement of approximately 5 Å). Trp129 and His145 clamp the quaternary ammonium moiety of choline and also connect the catalytic cleft to the C-terminus of an adjacent protomer. The structural information reported here contrasts with the proposed role of conformational dynamics in promoting the enzymatic catalytic proficiency of an enzyme.

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Crystal structures of human ENPP1 in apo and bound forms

Cited by 25 publications

References 35 publications

AlphaFill: enriching the AlphaFold models with ligands and co-factors

AlphaFill: enriching the AlphaFold models with ligands and co-factors

Probing pathophysiology of extracellular cGAMP with substrate-selective ENPP1

Structural insights into choline-O-sulfatase reveal the molecular determinants for ligand binding

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