An osteocalcin-deficient mouse strain without endocrine abnormalities.

Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, µCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc-allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution.

Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states.

Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (Vm or Vi-o). These transjunctional voltage dependent processes have been termed Vj- or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980's, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use the resulting information to derive a consensus atomic structure that is then subjected to rigorous validation. In this paper, we summarize our efforts to obtain and validate atomic models of the open and voltage-driven closed states of undocked connexin hemichannels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Structural studies of N-terminal mutants of Connexin 26 and Connexin 32 using (1)H NMR spectroscopy.

Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane. Using (1)H NMR we show the N-terminus of a syndromic deafness mutation Cx26G12R, producing "leaky channels", contains a turn around G12 which is less structured and more flexible than wild-type. In contrast, the N-terminal structure of the same mutation in Cx32 chimera, Cx32*43E1G12R shows a larger constricted turn and no membrane current expression but forms membrane inserted hemichannels. Their function was rescued by formation of heteromeric channels with wild type subunits. We suggest the inflexible Cx32G12R N-terminus blocks ion conduction in homomeric channels and this channel block is relieved by incorporation of wild type subunits. In contrast, the increased open probability of Cx26G12R hemichannels is likely due to the addition of positive charge in the channel pore changing pore electrostatics and impairing hemichannel regulation by Ca(2+). These results provide mechanistic information on aberrant channel activity observed in disease.

Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.

Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ßßα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ßßα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ßßα zinc finger domain, the C-terminal finger of the Wilms' tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)-WT1-4 followed by fluorescence spectroscopy yielded the effective pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) M(-1), with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least -17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)-WT1-4 to those from the model peptide Zn(II)-GGG-Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent Kf(ML) values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ≤+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, -17.6 kcal/mol.

The carboxyl terminal residues 220-283 are not required for voltage gating of a chimeric connexin32 hemichannel.

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of Vj- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the Vj (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and Vj gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32(∗)43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both Vj- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR(219)) of the Cx32(∗)43E1 hemichannel. We conclude that an operational Cx32 Vj (fast) gate does not require CT residues 220-283, as reported previously by others.

X-ray crystal structure of bovine 3 Glu-osteocalcin.

The 3 Glu form of osteocalcin (3 Glu-OCN) is increased in serum during low vitamin K intake or oral anticoagulant use (warfarin). Previous reports using circular dichroism show it is less structured than 3 Gla Ca²âº-osteocalcin and does not bind strongly to bone mineral. Recent studies have suggested a role for 3 Glu-OCN as a potential regulator of glucose metabolism. A G-protein-coupled receptor, GPRC6a, found in the pancreas and testes was identified as the putative osteocalcin receptor. The purpose of this study is to determine the high-resolution structure of bovine 3 Glu-OCN, using X-ray crystallography, to understand molecular interactions with mineral and the GPRC6a receptor. Diffraction quality crystals of thermally decarboxylated bovine osteocalcin were grown, and the crystal structure was determined to 1.88 Å resolution. The final refined structure contained residues 17-47 and, like 3 Gla Ca²âº-OCN, consisted of three α-helices surrounding a hydrophobic core, a C23-C29 disulfide bond between two of the helices, and no bound Ca²âº. Thus, the helical structure of 3 Glu-OCN is Ca²âº-independent but similar to that of 3 Gla Ca²âº-OCN. A reduced level of mineral binding could result from a lower number of Ca²âº coordinating ligands on 3 Glu-OCN. The structure suggests the GPRC6a receptor may respond to helical osteocalcin and will aid in providing molecular mechanistic insight into the role of 3 Glu-OCN in glucose homeostasis.

Progesterone-induced changes in the phosphoryl potential during the meiotic divisions in amphibian oocytes: role of Na/K-ATPase.

BACKGROUND: Progesterone triggers resumption of the first meiotic division in the Rana pipiens oocyte by binding to the N-terminal external loop of the catalytic subunit of Na/K-ATPase, releasing a cascade of lipid second messengers. This is followed by internalization of specific membrane proteins, plasma membrane depolarization and nuclear membrane breakdown, culminating in arrest at second metaphase. RESULTS: Progesterone initiates an increase in phosphoryl potential during the first meiotic division, resulting in the accumulation of high energy protein phosphate by second metaphase arrest. 31P-NMR, with saturation transfer, demonstrates that the phosphocreatine level rises ~2 fold and that the "pseudo" first order rate constant for the creatine kinase reaction falls to ~20% of the control by the onset of nuclear membrane breakdown. 32PO4 pulse-labeling reveals a net increase in phosphorylation of yolk protein phosvitin during this period. The increased yolk protein phosphorylation coincides with internalization of membrane Na/K-ATPase and membrane depolarizatio CONCLUSIONS: These results indicate that progesterone binding to the catalytic subunit of the Na-pump diverts ATP from cation regulation at the plasma membrane to storage of high energy phosphate in yolk protein. Phosvitin serves as a major energy source during fertilization and early cleavage stages and is also a storage site for cations (e.g. Na+, K+, Ca2+, Fe2+/3+) essential for embryonic development.

NMR and structural data for Connexin 32 and Connexin 26 N-terminal peptides.

In this article we present 1H and 13C chemical shift assignments, secondary structural propensity data and normalized temperature coefficient data for N-terminal peptides of Connexin 26 (Cx26), Cx26G12R and Cx32G12R mutants seen in syndromic deafness and Charcot Marie Tooth Disease respectively, published in "Structural Studies of N-Terminal Mutants of Connexin 26 and Connexin 32 Using 1H NMR Spectroscopy" (Y. Batir, T.A. Bargiello, T.L. Dowd, 2016) [1]. The mutation G12R affects the structure of both Cx26 and Cx32 peptides differently. We present data from secondary structure propensity chemical shift analysis which calculates a secondary structure propensity (SSP) score for both disordered or folded peptides and proteins using the difference between the 13C secondary chemical shifts of the Cα and Cß protons. This data supplements the calculated NMR structures from NOESY data [1]. We present and compare the SSP data for the Cx26 vs Cx26G12R peptides and the Cx32 and Cx32G12R peptides. In addition, we present plots of temperature coefficients obtained for Cx26, Cx26G12R and Cx32G12R peptides collected previously [1] and normalized to their random coil temperature coefficients, "Random coil 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG" (G. Merutka, H.J. Dyson, P.E. Wright, 1995) [2]. Reductions in these normalized temperature coefficients are directly observable for residues in different segments of the peptide and this data informs on solvent accessibility of the NH protons and NH protons which may be more constrained due to the formation of H bonds.

Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.