Koichiro Ishimori

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.

Molecular oxygen regulates the enzymatic activity of a heme-containing diguanylate cyclase (HemDGC) for the synthesis of cyclic di-GMP

We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O(2), but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O(2) sensing responsible for the regulation of the enzymatic activity.

A heme degradation enzyme, HutZ, from Vibrio cholerae

HutZ, one of the crucial proteins of the iron uptake system in Vibrio cholerae, was purified, which binds to heme at a stoichiometry of 1 : 1. In the presence of ascorbic acid, the HutZ-bound heme degrades via the same intermediates observed in heme oxygenase, suggesting that HutZ works as a heme degradation enzyme.

Conformational Disorder of the Most Immature Cu,Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.

Effects of the bHLH domain on axial coordination of heme in the PAS-A domain of neuronal PAS domain protein 2 (NPAS2): Conversion from His119/Cys170 coordination to His119/His171 coordination

Neuronal PAS domain protein 2 (NPAS2), which is a CO-dependent transcription factor, consists of a basic helix-loop-helix domain (bHLH), and two heme-containing PAS domains (PAS-A and PAS-B). In our previous study on the isolated PAS-A domain, we concluded that His119 and Cys170 are the axial ligands of the ferric heme, while Cys170 is replaced by His171 upon reduction of heme (Uchida et al., J. Biol. Chem. 270, (2005) 21358-21368.). Recently, we characterized the PAS-A domain combined with the N-terminal bHLH domain, and found that some spectroscopic features were different from those of the isolated PAS-A domain (Mukaiyama et al., FEBS J. 273, (2006) 2528-2539.). Therefore, we reinvestigated the coordination structure of heme in the bHLH-PAS-A domain and prepared four histidine and one cysteine mutants. Resonance Raman spectrum of the Cys170Ala mutant is the same as that of wild type with a dominant 6-coordinate heme in the ferric form. In contrast, His119Ala and His171Ala mutants significantly increase amounts of the 5-coordinate species, indicating that His119 and His171, not Cys170, are axial ligands of the ferric heme in the bHLH-PAS-A domain. We had confirmed that the coordination structure of the isolated PAS-A domain is in equilibrium between Cys-Fe-His and His-Fe-His coordinated species but newly found that interaction of the PAS-A domain with the bHLH domain shifts the equilibrium toward the latter structure. Such flexibility in the heme coordination structure seems to be in favor of signal transduction in NPAS2.

Redox-controlled backbone dynamics of human cytochrome c revealed by 15N NMR relaxation measurements.

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D 15N NMR relaxation experiments. 15N T1 and T2 values and 1H-15N NOEs of uniformly 15N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S2), the effective correlation time for internal motion (taue), the 15N exchange broadening contributions (Rex) for each residue, and the overall correlation time (taum) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S2 value was increased from 0.88+/-0.01 to 0.92+/-0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S2 values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.

Sequence and Temperature Dependence of the End-to-End Collision Dynamics of Single-Stranded DNA

Intramolecular collision dynamics play an essential role in biomolecular folding and function and, increasingly, in the performance of biomimetic technologies. To date, however, the quantitative studies of dynamics of single-stranded nucleic acids have been limited. Thus motivated, here we investigate the sequence composition, chain-length, viscosity, and temperature dependencies of the end-to-end collision dynamics of single-stranded DNAs. We find that both the absolute collision rate and the temperature dependencies of these dynamics are base-composition dependent, suggesting that base stacking interactions are a significant contributor. For example, whereas the end-to-end collision dynamics of poly-thymine exhibit simple, linear Arrhenius behavior, the behavior of longer poly-adenine constructs is more complicated. Specifically, 20- and 25-adenine constructs exhibit biphasic temperature dependencies, with their temperature dependences becoming effectively indistinguishable from that of poly-thymine above 335 K for 20-adenines and 328 K for 25-adenines. The differing Arrhenius behaviors of poly-thymine and poly-adenine and the chain-length dependence of the temperature at which poly-adenine crosses over to behave like poly-thymine can be explained by a barrier friction mechanism in which, at low temperatures, the energy barrier for the local rearrangement of poly-adenine becomes the dominant contributor to its end-to-end collision dynamics.

Amorphous Aggregation of Cytochrome C with Inherently low Amyloidogenicity is Characterized by the Metastability of Supersaturation and the Phase Diagram

Despite extensive studies on the folding and function of cytochrome c, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome c, metal-bound cytochrome c, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome c was low due to high solubility, markedly unfolded apo cytochrome c, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome c increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome c by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome c variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome c, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome c is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation.

Spectroscopic studies on HasA from Yersinia pseudotuberculosis

Heme acquisition system A (HasA) is known as a hemophore in Gram-negative pathogens. The ferric heme iron is coordinated by Tyr-75 and His-32 in holo-HasA from Pseudomonas aeruginosa (HasApa). In contrast, in holo-HasA from Yersinia pseudotuberculosis (HasAyp), our spectroscopic studies suggest that only Tyr-75 coordinates to the ferric heme iron. The substitution of Gln-32 with alanine in HasAyp does not alter the spectroscopic properties, indicating that Gln-32 is not an axial ligand for the heme iron. Somewhat surprisingly, the Y75A mutant of HasAyp can capture a free hemin molecule but the rate of hemin uptake is slower than that of wild type, suggesting that the hydrophobic interaction in the heme pocket may also play a role in heme acquisition. Unlike in wild type apoprotein, ferric heme transfer from Hb to Y75A apo-HasAyp has not been observed. These results imply that coordination (bonding/interaction) between Tyr-75 and the heme iron is important for heme transfer from Hb. Interestingly, HasAyp differs from HasApa in its ability to bind the ferrous heme iron. Apo-HasAyp can capture ferrous heme and resonance Raman spectra of ferrous-carbon monoxide holo-HasAyp suggest that Tyr-75 is protonated when the heme iron is in the ferrous state. The ability of HasAyp to acquire the ferrous heme iron might be beneficial to Y. pseudotuberculosis, a facultative anaerobe in the Enterobacteriaceae family.

Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation.

Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271–12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.