Structural characterization of anti-CRISPR AcrIIC4 from prophages of Haemophilus parainfluenza
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The CRISPR-Cas system employs RNA- guided endonucleases that recognize and cleave invading foreign nucleic acids. Phages produce anti- CRISPR proteins that inhibit target Cas proteins and facilitate subsequent infection. AcrIIC4 was identified from prophages of Haemophilus parainfluenza and potently inhibits type II-C Cas9. Previous crystal structures reported two distinct helical conformations of AcrIIC4. Here, we measured residual dipolar coupling constants of amide resonances to determine the conformational state of AcrIIC4 in solution. Our results unambiguously illustrate that AcrIIC4 forms a three-helical bundle in solution at neutral pH, which is maintained in the AcrIIC4-Cas9 complex. We also report the backbone resonance assignments of AcrIIC4.
Abstract
The CRISPR-Cas system employs RNA- guided endonucleases that recognize and cleave invading foreign nucleic acids. Phages produce anti- CRISPR proteins that inhibit target Cas proteins and facilitate subsequent infection. AcrIIC4 was identified from prophages of Haemophilus parainfluenza and potently inhibits type II-C Cas9. Previous crystal structures reported two distinct helical conformations of AcrIIC4. Here, we measured residual dipolar coupling constants of amide resonances to determine the conformational state of AcrIIC4 in solution. Our results unambiguously illustrate that AcrIIC4 forms a three-helical bundle in solution at neutral pH, which is maintained in the AcrIIC4-Cas9 complex. We also report the backbone resonance assignments of AcrIIC4.