Computational de novo design, and characterization of an A(2)B(2) diiron protein.
Diiron proteins are found throughout nature and have a diverse range of functions; proteins in this class include methane monooxygenase, ribonucleotide reductase, Delta(9)-acyl carrier protein desaturase, rubrerythrin, hemerythrin, and the ferritins. Although each of these proteins has a very different overall fold, in every case the diiron active site is situated within a four-helix bundle. Additionally, nearly all of these proteins have a conserved Glu-Xxx-Xxx-His motif on two of the four helices with the Glu and His residues ligating the iron atoms. Intriguingly, subtle differences in the active site can result in a wide variety of functions. To probe the structural basis for this diversity, we designed an A(2)B(2) heterotetrameric four-helix bundle with an active site similar to those found in the naturally occurring diiron proteins. A novel computational approach was developed for the design, which considers the energy of not only the desired fold but also alternatively folded structures. Circular dichroism spectroscopy, analytical ultracentrifugation, and thermal unfolding studies indicate that the A and B peptides specifically associate to form an A(2)B(2) heterotetramer. Further, the protein binds Zn(II) and Co(II) in the expected manner and shows ferroxidase activity under single turnover conditions.
Department of Biochemistry and Biophysics, School of Medicine, The University of Pennsylvania, 1010 Stellar-Chance Bldg, 421 Curie Blvd, Philadelphia 19104-6059, USA.
Christopher M
CM
Summa
Michael M
MM
Rosenblatt
Jae-Kyoung
JK
Hong
James D
JD
Lear
William F
WF
DeGrado
D000595:Amino Acid Sequence; D001665:Binding Sites; D002570:Ceruloplasmin / Q000378:metabolism; D002942:Circular Dichroism; D003035:Cobalt / Q000378:metabolism; D019295:Computational Biology / Q000379:methods*; D007501:Iron / Q000378:metabolism*; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D021122:Protein Subunits; D016415:Sequence Alignment; D013696:Temperature; D014461:Ultracentrifugation
D008667:Metalloproteins; D010455:Peptides; D021122:Protein Subunits; D003035:Cobalt; D007501:Iron; D002570:Ceruloplasmin