Tianlei Ying

Evolutionary alkaline transition in human cytochrome c

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pKa value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.

Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide

Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a α-subunit (690 AA) and a heme-binding β-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC β1 subunit (HsGCβ619) and its truncated N-terminal fragments (HsGCβ195 and HsGCβ384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV–vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC.

Structural Basis for Cytochrome c Y67H Mutant to Function as a Peroxidase

The catalytic activity of cytochrome c (cyt c) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe3+ ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H2O2 by functioning as an acidic catalyst.

A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase.

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV–vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k1 but a larger k2. Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.

Conformational Toggling of Yeast Iso-1-Cytochrome c in the Oxidized and Reduced States

To convert cyt c into a peroxidase-like metalloenzyme, the P71H mutant was designed to introduce a distal histidine. Unexpectedly, its peroxidase activity was found even lower than that of the native, and that the axial ligation of heme iron was changed to His71/His18 in the oxidized state, while to Met80/His18 in the reduced state, characterized by UV-visible, circular dichroism, and resonance Raman spectroscopy. To further probe the functional importance of Pro71 in oxidation state dependent conformational changes occurred in cyt c, the solution structures of P71H mutant in both oxidation states were determined. The structures indicate that the half molecule of cyt c (aa 50–102) presents a kind of “zigzag riveting ruler” structure, residues at certain positions of this region such as Pro71, Lys73 can move a big distance by altering the tertiary structure while maintaining the secondary structures. This finding provides a molecular insight into conformational toggling in different oxidation states of cyt c that is principle significance to its biological functions in electron transfer and apoptosis. Structural analysis also reveals that Pro71 functions as a key hydrophobic patch in the folding of the polypeptide of the region (aa 50–102), to prevent heme pocket from the solvent.

Generation of novel functional metalloproteins via hybrids of cytochrome c and peroxidase

The continued interest in protein engineering has led to intense efforts in developing novel stable enzymes, which could not only give boost to industrial and biomedical applications, but also enhance our understanding of the structure-function relationships of proteins. We present here the generation of three hybrid proteins of cytochrome c (cyt c) and peroxidase via structure-based rational mutagenesis of cyt c. Several residues (positions 67, 70, 71 and 80) in the distal heme region of cyt c were mutated to the highly conserved amino acids in the heme pocket of peroxidases. The multiple mutants were found to exhibit high peroxidase activity and conserve the impressive stability of cyt c. We expect that this strategy could be extended to other cases of metalloprotein engineering, and lead to the development of stable and active biocatalysts for industrial uses. Besides, this study also provides insight into the structure-function relationships of hemoproteins.

A route to novel functional metalloproteins via hybrids of cytochrome P450 and cytochrome c.

One challenging topic in the field of protein engineering is to design novel functional proteins, which would provide not only a critical test of our understanding of the structure–function relationships of proteins, but also applications towards biocatalysis, pharmaceuticals and diagnostics. Metalloproteins, which account for nearly half of all proteins in nature, are particularly attractive in engineering because of their ubiquitous presence in vivo such as in biocatalysis, in energy metabolism and in signal transduction. 2] Significant advances have been made in this area and, as recently reviewed by Lu, novel metalloproteins have mainly been designed by three strategies : the de novo design of protein scaffolds, the design of metal-binding sites in native scaffolds and the design of metalloproteins with the use of unnatural amino acids or non-native metal cofactors. Recently, we have been exploring a new route to novel functional metalloproteins, involving the construction of hybrid protein scaffolds from different metalloproteins. The hybrid scaffolds contain specific motifs as a result of rational design, and the resulting proteins combine the merits of distinct metalloproteins. Here we report the construction of five hybrid proteins through hybridization of two different metalloproteins—cytochrome c (Cyt c) and cytochrome P450 (CYP450)—by genetic and protein engineering. The gene overlap extension method was employed to construct the plasmids and a high-level expression and purification system for the hybrid proteins was developed. The stabilities and catalytic activities of the hybrid proteins were examined in detail. Interestingly, all the hybrid proteins possess superior stabilities, and some of the resulting metalloproteins have acquired the epoxidation activity of CYP450. More strikingly, the peroxidase activity of one particular hybrid protein is more than 40 times greater than that of Cyt c. One of the fundamental purposes of protein engineering is to develop stable enzymes for industrial applications. In this context, Cyt c, which has a remarkable stability attributed to its compact globular structure and covalently bound heme group, could serve as an excellent structural framework in engineering. CYP450, another heme-containing protein, is one of the most versatile enzymes in nature and could potentially efficiently catalyze hundreds of different substrates through a variety of difficult biotransformations such as epoxidations and hydroxylations. However, strict reaction conditions are required for CYP450 catalysis in vitro, obstructing possible industrial applications. In CYP450, six flexible protein segments, named “substrate recognition sites” (SRSs), are considered to be the key contributors to its catalytic diversity, involved in moving, recognizing and binding substrates in an induced-fit mechanism. 4] Thus, in order to obtain stable and catalytic proteins, the SRS segments of CYP450 were introduced into the scaffold of yeast iso-1 Cyt c by replacement of the residues in its 78–85 loop, which is one of the least stable and last folding units in Cyt c. The hybrid proteins of Cyt c with CYP450s SRS-1, SRS-2, SRS-3, SRS-5 and SRS-6 were named HY1, HY2, HY3, HY5 and HY6, respectively. Firstly, we conducted molecular dynamics simulations for the rational design of hybrid proteins. Whereas Cyt c does not have any cavity around the heme pocket, according to the analysis, it is interesting to note that HY1 and HY6 each possess a cavity in the distal heme region. In HY1 the volume of the pocket is over 150 3 (Figure 1), providing a molecular basis for the binding of substrates.

Manganese superoxide dismutase from human pathogen Clostridium difficile

Clostridium difficile is a human pathogen that causes severe antibiotic-associated Clostridium difficile infection (CDI). Herein the MnSODcd from C. difficile was cloned, expressed in Escherichia Coli,and characterized by X-ray crystallography, UV/Vis and EPR spectroscopy, and activity assay, et al. The crystal structure of MnSODcd (2.32xa0Å) reveals a manganese coordination geometry of distorted trigonal bipyramidal, with His111, His197 and Asp193 providing the equatorial ligands and with His56 and a hydroxide or water forming the axial ligands. The catalytic activity of MnSODcd (8,600xa0U/mg) can be effectively inhibited by 2-methoxyestradiol with an IC50 of 75xa0μM. The affinity investigation between 2-methoxyestradiol and MnSODcd by ITC indicated a binding constant of 8.6xa0μM with enthalpy changes (ΔHxa0=xa0−4.08xa0±xa00.03xa0kcal/mol, ΔSxa0=xa09.53xa0±xa00.02xa0cal/mol/deg). An inhibitory mechanism of MnSODcd by 2-methoxyestradiol was probed and proposed based on molecular docking models and gel filtration analysis. The 2-methoxyestradiol may bind MnSODcd to interfere with the cross-linking between the two active sites of the dimer enzyme, compromising the SOD activity. These results provide valuable insight into the rational design of MnSODcd inhibitors for potential therapeutics for CDI.