The zinc-binding motif in tankyrases is required for the structural integrity and proper function of the catalytic domain

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and they are therefore possible drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyl-transferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on activity, stability and structure of human tankyrases. We generated mutants of human TNKS1 and TNKS2 abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive likely due to introduction of major structural defects, the TNKS2 mutant remained active and displayed a different activity profile compared to the wild type.

The FtsHi Enzymes of Arabidopsis thaliana: Pseudo-Proteases with an Important Function

FtsH metalloproteases found in eubacteria, animals, and plants are well-known for their vital role in the maintenance and proteolysis of membrane proteins. Their location is restricted to organelles of endosymbiotic origin, the chloroplasts, and mitochondria. In the model organism Arabidopsis thaliana, there are 17 membrane-bound FtsH proteases containing an AAA+ (ATPase associated with various cellular activities) and a Zn2+ metalloprotease domain. However, in five of those, the zinc-binding motif HEXXH is either mutated (FtsHi1, 2, 4, 5) or completely missing (FtsHi3), rendering these enzymes presumably inactive in proteolysis. Still, homozygous null mutants of the pseudo-proteases FtsHi1, 2, 4, 5 are embryo-lethal. Homozygous ftshi3 or a weak point mutant in FTSHi1 are affected in overall plant growth and development. This review will focus on the findings concerning the FtsHi pseudo-proteases and their involvement in protein import, leading to consequences in embryogenesis, seed growth, chloroplast, and leaf development and oxidative stress management.

The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

The activity of the TRPM7 channel is negatively regulated by intracellular Mg2+. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg2+. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H2O2). Site-directed mutagenesis of full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. Mutation of C1809 or C1813 prevented expression of full-length TRPM7 on the plasma membrane. We therefore developed an assay to functionally reconstitute full-length TRPM7 by coexpressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than that of full-length TRPM7 and was insensitive to oxidative stress. Coexpression of M7cd and M7kd attenuated the inhibition by intracellular Mg2+ and restored sensitivity to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. We observed a similar effect when M7cd was coexpressed with the kinase-inactive mutant M7kd-K1645R, suggesting that the kinase activity is not essential for the reconstitution. However, coexpression of M7cd and M7kd carrying a mutation at either C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was observed when using M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by its kinase domain and that the cysteines in the zinc-binding motif play a role in the oxidative stress response of TRPM7.

Identification of MMP-like protein from Streptococcus mitis

Streptococcus mitis is a normal commensal of the human oral cavity and oropharynx. This microorganism is an opportunistic pathogen in immune compromised hosts and a cause of invasive diseases such as infective endocarditis. We isolated a matrix metalloprotease (MMP)-like protein of S. mitis. The gene for the MMP-like protein was found on the genome sequence database of S. mitis . The gene encodes a protein consisting of 240 amino acid residues with a conserved zinc-binding motif (HEXXHXXGXXH) among the matrix metallopeptidase family. The gene was PCR-amplified from S. mitis ATCC 49456 and cloned for construction of the recombinant protein. The recombinant MMP-like protein with a GST-tag was purified and the enzymatic activity was assessed. The recombinant protein showed approximately half the level of MMP-like activity as compared to human recombinant MMP-8. The MMP-like protein of S. mitis may be involved in the pathogenesis of infection to the dentin with collagen fibers and systemic invasive diseases.

Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

TRPM7 channel activity is negatively regulated by intracellular Mg2+. We previously reported that TRPM7 was inhibited by oxidative stress due to an enhancement of the inhibition by intracellular Mg2+. In the present study, we aimed to clarify the precise mechanism underlying the TRPM7 inhibition by oxidative stress induced by hydrogen peroxide (H2O2). Site-directed mutagenesis on full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain was involved in the H2O2-induced TRPM7 inhibition. When C1809 or C1813 was mutated, full-length TRPM7 failed to be expressed on the plasmamembrane. We therefore developed a novel approach in which the full-length TRPM7 is functionally reconstituted by co-expressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate individual proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ stronger than in full-length TRPM7. Co-expression of M7cd and M7kd attenuated the current inhibition by intracellular Mg2+, and the current was sensitive to oxidative stress, indicating a successful reconstitution of a full-length TRPM7-like current. A similar current reconstitution was observed when M7cd co-expressed with the kinase inactive mutant M7kd-K1645R. Thus, it was suggested that the kinase activity might not be essential for the reconstitution. Co-expression of M7cd and M7kd carrying a mutation at C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was also observed with M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation together with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by M7kd, and the cysteines in the zinc-binding motif might play a role in the oxidative stress response of TRPM7.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

First Crystal Structure of a Nonstructural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein

ABSTRACT Hepatitis E virus (HEV) is a 7.2-kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF1 to ORF3. All nonstructural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression, and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a nonclassical, potentially catalytic zinc-binding motif. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome. We present analysis for the first time of this identified nonstructural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology. IMPORTANCE Hepatitis E virus (HEV) is an emerging virus found predominately in developing countries; it causes an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the nonstructural proteins of HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. Here we present the first structural and biophysical characterization of an HEV nonstructural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease.

First Crystal Structure of a Non-Structural Hepatitis E Viral Protein Identifies a Putative Novel Zinc-Binding Protein

Hepatitis E virus (HEV) is a 7.2 kb positive-sense, single-stranded RNA virus containing three partially overlapping reading frames, ORF 1-3. All non-structural proteins required for viral replication are encoded by ORF1 and are transcribed as a single transcript. Computational analysis of the complete ORF1 polyprotein identified a previously uncharacterized region of predicted secondary structure bordered by two disordered regions coinciding partially with a region predicted as a putative cysteine protease. Following successful cloning, expression and purification of this region, the crystal structure of the identified protein was determined and identified to have considerable structural homology to a fatty acid binding domain. Further analysis of the structure revealed a metal binding site, shown unambiguously to specifically bind zinc via a non-classical, potentially catalytic zinc-binding motif. We present analysis for the first time of this identified non-structural protein, expanding the knowledge and understanding of the complex mechanisms of HEV biology.ImportanceHepatitis E virus (HEV) is an emerging virus found predominately in developing countries causing an estimated 20 million infections, which result in approximately 57,000 deaths a year. Although it is known that the non-structural proteins of the HEV ORF1 are expressed as a single transcript, there is debate as to whether ORF1 functions as a single polyprotein or if it is processed into separate domains via a viral or endogenous cellular protease. In the following paper, we present the first structural and biophysical characterization of a HEV non-structural protein using a construct that has partially overlapping boundaries with the predicted putative cysteine protease. Based on the structural homology of the HEV protein with known structures, along with the presence of a catalytic zinc-binding motif, it is possible that the identified protein corresponds to the HEV protease, which could require activation or repression through the binding of a fatty acid. This represents a significant step forward in the characterization and the understanding of the molecular mechanisms of the HEV genome.

Conformational dynamics and allosteric effect modulated by the unique zinc-binding motif in class IIa HDACs

Class IIa histone deacetylases (HDACs) have been considered as potential targets for the treatment of several diseases.