Terry L. Dowd

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.

The effect of Pb2+ on the structure and hydroxyapatite binding properties of osteocalcin

Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb(2+) at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb(2+) on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb(2+) induces a similar structure in osteocalcin as Ca(2+) but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb(2+) to osteocalcin (K(d)=0.085 microM) than Ca(2+) (K(d)=1.25 mM). The hydroxyapatite binding assays show that Pb(2+) causes an increased adsorption to hydroxyapatite, similar to Ca(2+), but at 2-3 orders of magnitude lower concentration. Low Pb(2+) levels (1 microM) in addition to physiological Ca(2+) levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca(2+) alone. These results suggest a molecular mechanism of Pb(2+) toxicity where low Pb(2+) levels can inappropriately perturb Ca(2+) regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb(2+)-intoxicated animals.

Low extracellular magnesium induces intracellular free Mg deficits, ischemia, depletion of high-energy phosphates and cardiac failure in intact working rat hearts: A 31P-NMR study

Hemodynamic and 31P-NMR spectroscopic studies were performed on intact, perfused working rat hearts exposed to low (0.3 mM) extracellular Mg([Mg2+]o). Low [Mg2+]o perfusion resulted in rapid and significant falls in cardiac output, coronary flow, stroke volume, developed pressure and the rate-pressure product. Concomitant with this O2 consumption decreased and lactate production increased. Hearts perfused with 0.3 mM, instead of 1.2 mM, [Mg2+]o exhibited significant reductions in [ATP], [PCr], intracellular free Mg ([Mg2+]i), and pHi; a marked rise in intracellular Pi corresponding to a precipitous fall in the cytosolic phosphorylation potential was seen. Reintroduction of 1.2 mM [Mg2+]o failed to reestablish either normal hemodynamics, or high-energy phosphates and intracellular Pi, suggesting irreversible myocyte injury. These observations are consistent with the tenet that low [Mg2+]o can result in marked reduction in oxygen and substrate delivery to the cardiac myocytes, probably as a result of coronary vasoconstriction.

The displacement of calcium from osteocalcin at submicromolar concentrations of free lead

Lead, an environmental toxin, is known to impair some of the functional properties of osteocalcin, a small protein (MW, 5700) active in bone mineralization and resorption. To investigate a possible mechanism of lead toxicity at the molecular level, we have studied the interaction of lead with osteocalcin using 43Ca and 1H NMR. The measured 43Ca NMR linewidth as well as longitudinal relaxation rate (1/T1) of 43CaCl2 progressively increased with increasing amounts of added osteocalcin. A titration measuring 43Ca linewidth as a function of [Ca2+]/[Osteocalcin] ratio could be fitted to a single metal binding site with a dissociation constant of 7 microM. The 43Ca 1/T1 of Ca-osteocalcin decreased in the presence of Pb2+ due to competitive displacement of Ca2+ by Pb2+. The magnitude of decrease in the effect of osteocalcin on 43Ca 1/T1 in the presence of Pb2+ was consistent with the existence of only one tight divalent cation binding site. An analysis of the NMR T1 data in osteocalcin solutions containing both Pb2+ and Ca2+ yielded a Pb-osteocalcin dissociation constant of about 2 nM. The 1H NMR spectra showed Pb-induced changes in the same aliphatic and aromatic resonances of osteocalcin that are also affected by Ca(2+)-binding, supporting interaction of Pb2+ at the Ca2+ site. However, the existence of significant differences between the Pb-osteocalcin and Ca-osteocalcin NMR spectra indicates some differences in the structures of the two complexes. Since Pb2+ inhibits the binding of osteocalcin to hydroxyapatite, the high affinity of Pb2+ for osteocalcin would indicate significant inactivation of osteocalcin even at submicromolar free lead levels. Pb(2+)-induced inactivation of osteocalcin could affect bone mineral dynamics and may be related to the observed inverse correlation between blood Pb(2+)-levels and stature and chest circumference observed in growing children.

The effect of lead on bone mineral properties from female adult C57/BL6 mice

Lead toxicity is a significant problem in the U.S. with elevated blood lead levels being highest among very young children and older adults >50 years old. Bone is the major reservoir of body lead, accounting for 75% in children and 90% in adults. Very little is known about the effect of lead on bone mineral properties in adults. We investigated the effect of lead on the femora from adult, 6 month old female C57/BL6 mice who were administered lead in the drinking water (250 ppm, blood lead 33 μg/dL) for 4 months. Bone mineral properties were examined using Fourier Transform Infrared Microscopy (FTIRM), quantitative microcomputed tomography (microCT) and whole bone mechanical testing. Lead significantly decreased the bone mineral density in the cortical and proximal cancellous bone and increased the marrow area in the cortical bone with microCT. Whole bone three-point bending showed a trend of decreased maximum and failure moments in the lead treated bones compared to controls. Lead significantly decreased the mineral/matrix ratio, collagen maturity and crystallinity in the trabecular bone as measured by FTIRM. In the cortical bone lead significantly decreased collagen maturity and bone crystal size by FTIRM. In contrast to cell culture studies, lead significantly increased serum osteocalcin levels. Lead also significantly increased the bone formation and resorption markers suggesting increased bone turnover. These data show that lead increases bone turnover resulting in weaker cortical bone in adult female mice and suggest that lead may exacerbate bone loss and osteoporosis in the elderly.

Structural studies of the N-terminus of Connexin 32 using 1H NMR spectroscopy

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of a representative connexin molecule contains a flexible turn around glycine 12 [P.E. Purnick, D.C. Benjamin, V.K. Verselis, T.A. Bargiello, T.L. Dowd, Arch. Biochem. Biophys. 381 (2000) 181-190] allowing the N-terminus to reside at the cytoplasmic entry of the channel forming a voltage-sensor. Previous functional studies or neuropathies have shown that the mutation G12Y and G12S form non-functional channels while functional channels are formed from G12P. Using 2D (1)H NMR we show that similar to G12, the structure of the G12P mutant contains a more flexible turn around residue 12, whereas the G12S and G12Y mutants contain tighter, helical turns in this region. These results suggest an unconstrained turn is required around residue 12 to position the N-terminus within the pore allowing the formation of the cytoplasmic channel vestibule, which appears to be critical for proper channel function.

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 ββα 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 ββα 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/ββα 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 pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)–WT1-4, KfML value of 7.5 × 1012 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 KfML 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.

Structural studies of N-terminal mutants of Connexin 32 using 1H NMR spectroscopy

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of functional connexin molecules contains a flexible turn around G12 (Arch. Biochem. Biophys.490:9,2009) allowing the N-terminus to form a portion of the channel pore near the cytoplasmic entrance. The mutants of nonfunctional connexin molecules G12S and G12Y were found to prevent this turn. Previous functional studies of loci at which Cx32 mutations cause a peripheral neuropathy, Charcot-Marie-Tooth disease, have shown that G12S is not plasma membrane inserted. Presently, we solve the structure of nonfunctional Connexin 32 mutants W3D and Y7D which do not appear to be membrane inserted. Using 2D (1)H NMR, we report that similar to G12S and G12Y, alterations in hydrophobic sidechain interactions disrupt (Y7D) or constrain (W3D) the flexible turn around G12. The alteration in the open turn around residue 12, observed in all nonfunctional mutants G12S, G12Y, W3D and Y7D correlates with loss of function. We propose that loss of the open turn causes the N-terminus to extend out of the channel pore and this misfolding may target mutants for destruction in the endoplasmic reticulum.

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 Ca²⁺-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 Ca²⁺-OCN, consisted of three α-helices surrounding a hydrophobic core, a C23-C29 disulfide bond between two of the helices, and no bound Ca²⁺. Thus, the helical structure of 3 Glu-OCN is Ca²⁺-independent but similar to that of 3 Gla Ca²⁺-OCN. A reduced level of mineral binding could result from a lower number of Ca²⁺ 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.

19F-NMR study of the effect of lead on intracellular free calcium in human platelets

Lead has been shown to affect calcium homeostasis. However, there is no prior evidence to indicate an effect of low concentrations of lead in the environment (approximately 1 microM) on the intracellular free Ca2+ concentration in any human tissue. We have investigated the effect of lead on the intracellular free Ca2+ concentration of human blood platelets using 19F-NMR and a fluorinated intracellular Ca2+ indicator. We report a basal intracellular free Ca2+ value of 172 +/- 8 nM. Treatment with 1, 5, 10 and 25 microM Pb2+ resulted in average increases in intracellular free Ca2+ of 39%, 91%, 135% and 172%, respectively. The percent increase in intracellular free Ca2+ was linearly and positively correlated with the log of Pb2+ concentration. Using atomic absorption spectroscopy, a significant increase in total calcium of approx. 10 nmol/mg protein was found in 25 microM Pb2+ treated platelets. This indicates that influx of external Ca2+ contributes to the observed increase in free Ca2+. The results provide an explanation for the previously reported effects of lead on platelet function, and suggest a mechanism for low level lead-induced hypertension.