Lemak Sofia, Nocek Boguslaw, Beloglazova Natalia, Skarina Tatiana, Flick Robert, Brown Greg, Joachimiak Andrzej, Savchenko Alexei, Yakunin Alexander F
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL 60439, USA.
Nucleic Acids Res. 2014;42(17):11144-55. doi: 10.1093/nar/gku797. Epub 2014 Sep 8.
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.
Cas4核酸酶构成了CRISPR（成簇规律间隔短回文重复序列）相关蛋白的一个核心家族，但对其结构和活性了解甚少。在此，我们报道了来自嗜热栖热放线菌的Cas4蛋白Pcal_0546的晶体结构，该结构揭示了一种具有RecB样折叠的单体蛋白以及一个由四个保守半胱氨酸残基配位的[2Fe-2S]簇。Pcal_0546对单链DNA底物表现出金属依赖性的5'至3'核酸外切酶活性，而来自嗜酸热硫化叶菌的Cas4蛋白SSO1391可以在5'至3'和3'至5'两个方向上切割单链DNA。Pcal_0546的活性位点包含一个由Asp123、Glu136、His146的侧链以及Ile137的主链羰基配位的结合金属离子。对Pcal_0546和SSO1391进行定点诱变表明，RecB基序II、III和QhXXY的残基对核酸酶活性至关重要，而保守半胱氨酸残基的突变导致铁硫簇的丢失，但对DNA切割没有影响。我们的结果揭示了Cas4核酸酶的生化多样性，其可以具有不同的寡聚状态，包含[4Fe-4S]或[2Fe-2S]簇，并在不同方向上切割单链DNA产生单链DNA突出端，这些突出端是合成新的CRISPR间隔序列的潜在中间体。
