Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

AbstractApoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases require an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared to caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

Download Full-text

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Biochemical Journal ◽

10.1042/bcj20190625 ◽

2019 ◽

Vol 476 (22) ◽

pp. 3475-3492 ◽

Cited By ~ 6

Author(s):

Robert D. Grinshpon ◽

Suman Shrestha ◽

James Titus-McQuillan ◽

Paul T. Hamilton ◽

Paul D. Swartz ◽

...

Keyword(s):

Substrate Specificity ◽

Binding Pocket ◽

Gene Duplications ◽

Substrate Selection ◽

Charged Amino Acids ◽

Effector Caspase ◽

Effector Caspases ◽

Hydrophobic Binding ◽

Caspase 6 ◽

Aspartate Residue

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

Download Full-text

Caspases from Scleractinian Coral Show Unique Regulatory Features

10.1101/2020.04.13.039685 ◽

2020 ◽

Author(s):

Suman Shrestha ◽

Jessica Tung ◽

Robert D. Grinshpon ◽

Paul Swartz ◽

Paul T. Hamilton ◽

...

Keyword(s):

Binding Pocket ◽

Substrate Selection ◽

Amino Terminal ◽

Stony Coral ◽

Initiator Caspases ◽

Activation Mechanisms ◽

Immune Challenges ◽

Caspase 6 ◽

Biochemical Analyses ◽

Inflammatory Caspases

AbstractDiseases affecting coral have led to massive decline and altered the community structure of reefs. In response to immune challenges, cnidaria activate apoptotic or autophagic pathways, and the particular pathway correlates with disease sensitivity (apoptosis) or resistance (autophagy). Although cnidaria contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive stony coral, and Porites astreoides, a disease-resistant stony coral. The four caspases are predicted homologs of human caspases-3 and −7, but OfCasp3a and PaCasp7a contain an amino-terminal caspase activation and recruitment domain (CARD) similar to human initiator/inflammatory caspases. In contrast, OfCasp3b and PaCasp3 have short pro-domains, like human effector caspases. We show that OfCasp3a and PaCasp7a are DxxDases, like human caspases-3 and −7, while OfCasp3b and PaCasp3 are more similar to human caspase-6, with VxxDase activity. Our biochemical analyses suggest a mechanism in coral in which the CARD-containing DxxDase is activated on death platforms, but the protease does not directly activate the VxxDase. We also report the first X-ray crystal structure of a coral caspase, that of PaCasp7a determined at 1.57Å resolution. The structure reveals overall conservation of the caspase-hemoglobinase fold in coral as well as an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species, suggesting mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.

Download Full-text

Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket

ACS Synthetic Biology ◽

10.1021/acssynbio.9b00008 ◽

2019 ◽

Vol 8 (6) ◽

pp. 1325-1336 ◽

Cited By ~ 6

Author(s):

Naazneen Sofeo ◽

Jason H. Hart ◽

Brandon Butler ◽

David J. Oliver ◽

Marna D. Yandeau-Nelson ◽

...

Keyword(s):

Substrate Specificity ◽

Binding Pocket ◽

Acetyl Coa ◽

Rational Mutagenesis

Download Full-text

Negatively-charged amino acids at the peptide-binding pocket of HLA-DPB1 alleles are associated with susceptibility to anti-topoisomerase I-positive systemic sclerosis

Human Immunology ◽

10.1016/j.humimm.2016.05.012 ◽

2016 ◽

Vol 77 (7) ◽

pp. 550-554 ◽

Cited By ~ 2

Author(s):

Jeong Seok Lee ◽

Jin Kyun Park ◽

Heung Jae Kim ◽

Hyung Ki Lee ◽

Yeong Wook Song ◽

...

Keyword(s):

Amino Acids ◽

Systemic Sclerosis ◽

Topoisomerase I ◽

Peptide Binding ◽

Binding Pocket ◽

Charged Amino Acids

Download Full-text

Structure of the Lifeact–F-actin complex

PLoS Biology ◽

10.1371/journal.pbio.3000925 ◽

2020 ◽

Vol 18 (11) ◽

pp. e3000925 ◽

Cited By ~ 1

Author(s):

Alexander Belyy ◽

Felipe Merino ◽

Oleg Sitsel ◽

Stefan Raunser

Keyword(s):

Hypervariable Region ◽

Binding Pocket ◽

Actin Binding ◽

Filamentous Actin ◽

Activity Measurements ◽

High Resolution Structure ◽

D Loop ◽

Hydrophobic Binding

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

Download Full-text

Synthesis and Axial-Ligand Binding of Zinc Complexes of Amphiphilic Porphyrins Containing a Hydrophobic Binding Pocket

Chemistry Letters ◽

10.1246/cl.1997.819 ◽

1997 ◽

Vol 26 (8) ◽

pp. 819-820 ◽

Cited By ~ 3

Author(s):

Hiroyasu Imai ◽

Hiroki Munakata ◽

Atsuko Takahashi ◽

Shigeo Nakagawa ◽

Yoshio Uemori

Keyword(s):

Ligand Binding ◽

Binding Pocket ◽

Axial Ligand ◽

Zinc Complexes ◽

Hydrophobic Binding

Download Full-text

Characterization of calcium- and integrin-binding protein 1 (CIB1) knockout platelets: Potential compensation by CIB family members

Thrombosis and Haemostasis ◽

10.1160/th08-06-0351 ◽

2008 ◽

Vol 100 (05) ◽

pp. 847-856 ◽

Cited By ~ 19

Author(s):

Brenda R. Temple ◽

Holly R. Gentry ◽

Jan C. DeNofrio ◽

Weiping Yuan ◽

Leslie V. Parise

Keyword(s):

Thrombus Formation ◽

Mrna Level ◽

Family Members ◽

Cytoplasmic Tail ◽

Binding Pocket ◽

Vitro Binding ◽

Cytoplasmic Tails ◽

Hydrophobic Binding

SummaryPlatelet aggregation requires activation of the αIIbβ3 integrin,an event regulated by the integrin cytoplasmic tails. CIB1 binds to the cytoplasmic tail of the integrin αIIb subunit. Previous overexpression and knockdown studies in murine megakaryocytes demonstrated that CIB1 inhibits integrin αIIbβ3 activation.Here we analyzed Cib1-/- mice to determine the function of CIB1 in platelets in vitro and in vivo. We found that although these mice had no overt platelet phenotype, mRNA level of CIB1 homolog CIB3 was increased in Cib1-/- megakaryocytes. In vitro binding experiments showed that recombinant CIB1, -2 and -3 bound specifically to an αIIb cytoplasmic tail peptide. Subsequent protein modeling experiments indicated that CIBs 1–3 each have a highly conserved hydrophobic binding pocket. Therefore, the potential exists for compensation for the loss of CIB1 by these CIB family members, thereby preventing pathologic thrombus formation in Cib1-/- mice.

Download Full-text

Caspase-9 cleavage, do you need it?

Biochemical Journal ◽

10.1042/bj20070617 ◽

2007 ◽

Vol 405 (1) ◽

Cited By ~ 34

Author(s):

Davina Twiddy ◽

Kelvin Cain

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Caspase 9 ◽

Autocatalytic Cleavage ◽

Downstream Effector ◽

Apoptosis Inhibition ◽

Biochemical Journal ◽

Effector Caspase ◽

Effector Caspases

Caspase-9, which is activated by association with the Apaf-1 (apoptotic protease-activating factor-1) apoptosome complex, cleaves and activates the downstream effector caspases-3 and -7, thereby executing the caspase-cascade and cell-death programme. Although caspase-9 does not need to be cleaved to be active, apoptotic cell death is always accompanied by autocatalytic cleavage and by further downstream effector caspase-dependent cleavage of caspase-9. In this issue of the Biochemical Journal, Denault and co-workers evaluate the role of caspase-3-dependent cleavage of caspase-9 and conclude that this mechanism mainly serves to enhance apoptosis by alleviating XIAP (X-linked inhibitor of apoptosis) inhibition of the apical caspase.

Download Full-text

Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca2+-calmodulin

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798315021609 ◽

2016 ◽

Vol 72 (1) ◽

pp. 83-92 ◽

Cited By ~ 5

Author(s):

Jiusheng Lin ◽

Henry van den Bedem ◽

Axel T. Brunger ◽

Mark A. Wilson

Keyword(s):

Crystal Structures ◽

Electron Density ◽

Full Range ◽

Binding Pocket ◽

Data Set ◽

Conformational Substates ◽

Density Maps ◽

Hydrophobic Binding ◽

Ef Hands ◽

Critical Regions

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca2+-CaM, but no assignment of these features has been made. In addition, Ca2+-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca2+-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca2+-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca2+-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca2+-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca2+-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

Download Full-text