Mechanism of Zn2+ and Ca2+ Binding to Human S100A1

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 μm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

The S2–S3 Loop of Kv7.4 Channels Is Essential for Calmodulin Regulation of Channel Activation

Kv7.4 (KCNQ4) voltage-gated potassium channels control excitability in the inner ear and the central auditory pathway. Mutations in Kv7.4 channels result in inherited progressive deafness in humans. Calmodulin (CaM) is crucial for regulating Kv7 channels, but how CaM affects Kv7 activity has remained unclear. Here, based on electrophysiological recordings, we report that the third EF hand (EF3) of CaM controls the calcium-dependent regulation of Kv7.4 activation and that the S2–S3 loop of Kv7.4 is essential for the regulation mediated by CaM. Overexpression of the mutant CaM1234, which loses the calcium binding ability of all four EF hands, facilitates Kv7.4 activation by accelerating activation kinetics and shifting the voltage dependence of activation leftwards. The single mutant CaM3, which loses the calcium binding ability of the EF3, phenocopies facilitating effects of CaM1234 on Kv7.4 activation. Kv7.4 channels co-expressed with wild-type (WT) CaM show inhibited activation when intracellular calcium levels increase, while Kv7.4 channels co-expressed with CaM1234 or CaM3 are insensitive to calcium. Mutations C156A, C157A, C158V, R159, and R161A, which are located within the Kv7.4 S2–S3 loop, dramatically facilitate activation of Kv7.4 channels co-expressed with WT CaM but have no effect on activation of Kv7.4 channels co-expressed with CaM3, indicating that these five mutations decrease the inhibitory effect of Ca2+/CaM. The double mutation C156A/R159A decreases Ca2+/CaM binding and completely abolishes CaM-mediated calcium-dependent regulation of Kv7.4 activation. Taken together, our results provide mechanistic insights into CaM regulation of Kv7.4 activation and highlight the crucial role of the Kv7.4 S2–S3 loop in CaM regulation.

The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca2+-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca2+-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pKa of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca2+-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg2+-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca2+/Mg2+ affinity of the EF site, and accelerates Ca2+ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca2+ affinity.

Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A

Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

Functional and structural characterization of allosteric activation of Phospholipase Cε by Rap1A

ABSTRACTPhospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with small GTPases, including Rap1A and Ras. While Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability, or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors (β-ARs), translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation, and identified hydrophobic residues on the surface of the RA2 domain that are also necessary for activation by the GTPase. Finally, small angle X-ray scattering (SAXS) showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. This data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLC core.

Fungal Genomes: Suffering with Functional Annotation Errors

Background The genome sequencing data are accumulating at a rapid pace, with the current genome sequence data of more than 5780 species being publicly available at the National Center for Biotechnology Information (NCBI) database alone. However, for the researcher communities to use these data, an error-free functional annotation report is a must. Results Analyses of the whole proteome sequence data of 689 fungal species (7.15 million protein sequences) to find the presence of functional annotation error in several species. Hence, calcium dependent protein kinases (CDPKs) and selenoproteins were targeted for the analysis as it is absent all across the fungi kingdom. The analyses revealed the presence of protein with the functional annotation name CDPK. InterproScan analysis revealed that, none of the protein sequences tagged with name “calcium dependent protein kinase” was found to encode calcium binding EF-hands at the regulatory domain. Similarly, none of a protein sequences with annotation name associated with “selenocysteine” was found to encode Sec (U) amino acid. Conclusion The presence of naming of such functional annotation errors in the fungal kingdom is raised a great concern and need to address it at the earliest possible time.

The Mechanism of MICU-Dependent Gating of the Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ uniporter (MCU) mediates mitochondrial Ca2+ uptake, regulating ATP production and cell death. According to the existing paradigm, MCU is occluded at the resting cytosolic [Ca2+] and only opens above an ∼400 nM threshold. This Ca2+-dependent gating is putatively conferred by MICUs, EF hand-containing auxiliary subunits that block/unblock the MCU pore depending on cytosolic [Ca2+]. Here we provide the first direct, patch-clamp based analysis of the Ca2+-dependent MCU gating and the role played by MICUs. Surprisingly, MICUs do not occlude the MCU pore, and MCU is a constitutively active channel without cytosolic [Ca2+] activation threshold. Instead, MICUs potentiate MCU activity when cytosolic Ca2+ binds to their EF hands. MICUs cause this potentiation by increasing the probability of open state of the MCU channel.One Sentence SummaryAuxiliary MICU subunits do not occlude the mitochondrial Ca2+ uniporter (MCU) but increase its activity as cytosolic Ca2+ is elevated.

Structural and mechanistic insights into secretagogin-mediated exocytosis

Secretagogin (SCGN) is a hexa–EF-hand protein that is highly expressed in the pancreas, brain, and gastrointestinal tract. SCGN is known to modulate regulated exocytosis in multiple cell lines and tissues; however, its exact functions and underlying mechanisms remain unclear. Here, we report that SCGN interacts with the plasma membrane SNARE SNAP-25, but not the assembled SNARE complex, in a Ca2+-dependent manner. The crystal structure of SCGN in complex with a SNAP-25 fragment reveals that SNAP-25 adopts a helical structure and binds to EF-hands 5 and 6 of SCGN. SCGN strongly inhibits SNARE-mediated vesicle fusion in vitro by binding to SNAP-25. SCGN promotes the plasma membrane localization of SNAP-25, but not Syntaxin-1a, in SCGN-expressing cells. Finally, SCGN controls neuronal growth and brain development in zebrafish, likely via interacting with SNAP-25 or its close homolog, SNAP-23. Our results thus provide insights into the regulation of SNAREs and suggest that aberrant synapse functions underlie multiple neurological disorders caused by SCGN deficiency.