Structural Basis And Molecular Mechanism Underlying The Functions Of Some Key Metalloproteins

Metalloproteins are defined to be a group of proteins using metal ions or metal containing prosthetic functionalities as their cofactors. Among all the proteins, Metalloproteins have accounted alomst half quantity. Metalloproteins have accomplished versatile functions spanning metal homeostasis, electron transfer, oxidative stress response, gene regulation, signal transduction and matter/energy metabolism etc. Metalloproteins always are the key determinants in the, physiological/pathological processes, and their expressions, locations, distributions and structurally functional alterations are tightly associated with the transitions of physiology and pathology. Metalloproteins often display selectivity for their metal cofactors and the binding of the metal cofactor would induce the structural and functional modulations for metalloproteins. This thesis have study some key metalloproteins mediating the physiological and pathological processes with respect to their gene cloning, protein expressions and purifications and their structure-reaction-property-function relationships. The details are depicted as following:The first chapter have reviewed the structural features and biological functions of metalloproteins, the currently hot research fields for metalloproteins and the techniques and methods usually explored to study metalloproteins.The second and third chapter have systematically studied the structure, function, inhibition and the molecular mechanism underlying the metal specificity for C. difficile MnSOD (SODcd). C. difficile, an anaerobic antibiotics resistance spore-forming human pathogen, easily infects antibiotic associated patients and causes pseudomembranous colitis. We noted that the function of MnSOD is tightly associated with the virulence of C. difficile. In chapter 2, we cloned and expressed SODcd in E. coli, solved its crystal structure and measured its SOD activity for the first time. Furthermore, we explored the inhibitory effect of 2-methoxyestradiol (2-ME) on SODcd activity by using in silico protein-ligand docking and Isothermal Titration Calorimetry (ITC). We established that 2-ME binds into the MnSODcd dimer interface cleft with a dissociation constant of 8.6 μM, which could intervene the cross-link between the metal center of two SODcd monomer and exhibit inhibitory effect. In the experimental process, we found SODcd can be occupied by various kinds of metal ions, but only Mn2+ and Fe3+ are the effect cofactors while Co2+, Cu2+, Ni2+ substituted SODcd exhibited catalytic incompetence. Thus to maximize its function, SODcd needs to exquisitely discriminate the miscellaneous trace transition metal ions in the human digestive tract. To understand the molecular mechanism underlying this metal specificity for MnSODcd, in chapter 3 we combined crystallography, spectroscopy, electrochemistry and DFT computations methods to dissect the structure-function relationships of different metal ions occupied SODcd. Unexpectedly, the metal equilibrium dialysis coupled ICP/MS, Trp intrinsic fluorescence kinetics, CD thermal unfolding experiments showed SODcd have a strongest binding affinity tarwards Co2+ and Co2+ -sub-MnSODcd was thermodynamically most stable, which was also supported by the DFT derived metal substitution reaction free energy. To explore the metal reactivity for SODcd, we utilized protein denaturation methods to prepare pure metal ion, i.e. Mn2+, Fe3+, Co2+ occupied SODcd. The metal fidelities of which were characterized by ICP/MS, UV/Vis, EPR spectra. Interestingly, the XO/cyt c activity assay showed MnSODcd exhibited highest activity and Fe-sub-MnSODcd only 1/10 activity while Co-sub-MnSODcd displayed no detectable activity. The crystal structures of these metal occupied SODcd and DFT-NBO analysis indicated that although their overall structures and the active site metal coordination geometry are highly similar, substantial differences are located in the conserved hydrogen bond network consisted of Tyr60, Tyr64, Gln 178 and the coordinated solvent. The hydrogen bond strength of Glnl78-H2O follows:Co-sub-MnSODcd> Fe-sub-MnSODcd> MnSODcd while for Glnl78-Tyr64, the sequence contrary become: Co-sub-MnSODcd< Fe-sub-MnSODcd< MnSODcd. Since for SOD function, its optimal Em needs to intermediate between those of O2/O2- (-0.16 V) and O2-/H2O2 (0.89 V) (vs. NHE), the hydrogen bond of Glnl78-H2O could impact SOD activity via tuning the redox potentials of metal ions and Gln178-Tyr64 could modulate catalytic ability by mediating the substrate reorientation in the proximity of active site via Tyr64 hydrogen bonding to the substrate. Through the electrochemical and spectroscopic studies, we demonstrated that the active site of MnSODcd possesses optimal Em and highest substrate binding affinity for O2- disproportionation, which accounts for its highest activity. Based on our data, we concluded that the metal intrinsic characters (d electronic configurations and coordination principles) and the exquisite hydrogen bond network co-determine the metal specificity for SODcd, which is also reinforced by the DFT derived free energy analysis for reaction transition states. Our results would pave a solid way for developing effectively specific inhibitors retarding C. difficile associated disease.The forth chapter have studied the metal binding and metal dependent DNA binding for a metalloregulator (TroR) from human pathogen T. pallidum. T. pallidum is the causative agent of syphilis. The gene loci of this pathogen locates a metal dependent cis-tro operon termed troABCDR. TroR, a negative gene transcription factor, could be activated by the cytoplasmic high concentration metal ions and be achieved to a proper confirmation to target the palindrome sequence upstream the troABC gene, leading to the transcriptional depression of TroABC transporter and decreasing the extracellular metal extraction. TroR belongs to the DtxR regulator superfamily. The sequence alignment and phylogenic analysis indicate TroR bearing novel metal binding site is a evolutional intermediate in DtxR/MntR family, being reminiscent of its distinct metal binding characters. Compared to DtxR and MntR, TroR is poorly characterized regarding to metal ligation, subsequent conformation alterations and DNA binding because prokaryotic expression of soluble TorR was very difficult due to the inclusion body precipitation. Thus, we developed a novel "double tag" method to obtain successfully the soluble TroR in E. coli. The ICP/OES, ITC demonstrated that TroR could bind Co2+, Ni2+, Mn2+ but not Zn2+. Further, the CD, ANS fluorescence experiments indicated only Co2+ and Mn2+ could induce conformational change for TroR, which is a prerequisite for its cognate DNA binding. By the site-directed mutation study, we identified TroR N11 plays important roles in metal binding and conformation alteration. Finally we designed the EMSA, fluorescence anistropy and ITC experiments to explore the metal ions dependent DNA binding of TroR and found TroR is a Mn2+ dependent regulator.The chapter 5 have elegantly dissected the structural basis and molecular mechanism underlying β-amyloid precursor protein (APP) and Death Receptor 6 (DR6) mediating neuronal degenerations. In 2009, Nikolaev’s reported the NGF deprivation regulated the shedding of APP to produce the ～35 kD fragment (N-APP) to activate caspases via death receptor 6 (DR6) dependent manner and potentiated neuronal and axonal degeneration. We are dedicated to dissect the structural basis of N-APP/DR6 interaction; explore the effect of AD brain overloaded Cu2+, Zn2+on this interaction; the molecular mechanism underlying the neurotoxicity of N-APP. To achieve these goals, we combined a series of biophysical experiments including ITC, FRET, crystallography, SXAS, protein/protein docking to characterize the NAPP/DR6 interaction and evaluated the impaction of the AD overloaded Cu2+, Zn2+ on this interaction. The results showed APP18-286 hinges for DR6 Cys rich domain (CRD) via APP18-126 surface hydrophobic patch and APP108-286 (acid region) highly negatively charged tail. Cu+ and Zn+ could facilitate this interaction. The neuotoxicity assays on SH-SY5Y, PC 12 and the primary neurons demonstrated via a DR6 dependent manner, N-APP could activate caspase and meanwhile induce the production of ROS, both of which synergistically exacerbate neuronal body apoptosis and axonal pruning. The structurally proposed NAPP/DR6 interaction model and the NAPP neurotoxicity would be useful for the structure based antagonist development against the progressive of AD.It is reported APP E2 domain (E2) owned ferroxidase activity and mediated the intracellular Fe2+ export by interacting with ferropotin (FPN1), thus preventing intracellular Fe2+ precipitation and oxidative stress. However, Hagen et al. argued against the ferroxidase function of E2 by designing a series of elegant ferroxidase activity assays and they contended that E2 could not even bind Fe2+ via anaerobic ITC experiment. The iron covalent variations involve the cellular iron transport, storage, excretion and oxidative stress processes. In the chapter 6, we explored the roles of APP E2 on the cerebral iron homeostasis and the underlying molecular mechanism. The ICP/OES, glycine competition titrations, ITC, EPR demonstrated E2 could bind Fe3+ although it could not bind Fe2+. More importantly, E2 could low the redox potential of Fe3+/Fe2+from 0.76 V to 0.28 V (vs. NHE), which may have biological implications considering that E2 prevents Fe+ from reducing, thus avoids the Fe2+ associated ROS production in CNS. On the other hand, E2 maybe benefits brain iron homeostasis by interacting with FPN1 and protecting it from degradation by proteasome (FPN1 may could later employ other ferroxidase partners to export the intracellular Fe2+, such as hephaestin). We prepared E2-FPN1HBD hybrid fusion protein to study the structure basis underlying E2 and FPN1 interaction.