HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 27, 18782-18791, July 4, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/27/18782    most recent M801400200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Igarashi, J. Articles by Shimizu, T. Search for Related Content PubMed PubMed Citation Articles by Igarashi, J. Articles by Shimizu, T. Social Bookmarking                      What's this?

Elucidation of the Heme Binding Site of Heme-regulated Eukaryotic Initiation Factor 2 Kinase and the Role of the Regulatory Motif in Heme Sensing by Spectroscopic and Catalytic Studies of Mutant Proteins^*

Jotaro Igarashi¹, Motohiko Murase¹, Aya Iizuka, Fabio Pichierri, Marketa Martinkova², and Toru Shimizu³

From the Institute of Multidisciplinary Research for Advanced Materials and Department of Applied Chemistry, Graduate School of Engineering, Tohoku University at Katahira, Sendai 980-8577, Japan

Heme-regulated eukaryotic initiation factor 2 (eIF2) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory Cys-Pro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.

Received for publication, February 21, 2008 , and in revised form, May 1, 2008.

^* This work was supported in part by Grants-in-aid for Scientific Research 19770099 (to J. I.) and 17101002 (to T. S.) and by the Special Education and Research Expenses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and a Postdoctoral Fellowship for Foreign Researchers from the Japan Society for the Promotion of Science P04110 [GenBank] (to M. Martinkova). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1S and Figs. 1S–8S.

¹ These authors contributed equally to this work.

² Present address: Dept. of Biochemistry, Faculty of Science, Charles University, Hlavova 2030/8, Prague 2, 128 40, Czech Republic.

³ To whom correspondence should be addressed: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Tel.: 81-22-217-5604 or -5605; Fax: 81-22-217-5604, -5605, or -5390; E-mail: shimizu{at}tagen.tohoku.ac.jp.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?