title: The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity.
abstract: Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5' to 3' exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5' to 3' and 3' to 5' directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Lemak
authors.affiliation: Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Nocek
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Natalia
authors.initials: N
authors.lastname: Beloglazova
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Skarina
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Flick
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Greg
authors.initials: G
authors.lastname: Brown
authors.affiliation: Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada a.iakounine@utoronto.ca.
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D041543:Pyrobaculum / Q000201:enzymology*; D048229:Sulfolobus solfataricus / Q000201:enzymology
chemical_list: D019843:Archaeal Proteins; D064130:CRISPR-Associated Proteins; D007506:Iron-Sulfur Proteins; D003851:Deoxyribonucleases