| The clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated(Cas)proteins establish the adaptive immunity system in prokaryotes to defend against invading phages and plasmids.Short DNA sequences from invading pathogens are integrated into the CRISPR locus as immunological memory,which is then transcribed and processed into mature cr RNA,Cas protein and cr RNA form interference complexes,and used to target and degrade the foreign DNAs.In the initial adaptation stage,the foreign DNA sequences are processed into prespacer,and the Cas1 and Cas2 protein complexes are responsible for binding prespacer and integrating it into the host genomic CRISPR locus,generating a new spacer within the CRISPR array.Cas1 and Cas2 form a structurally stable protein complex: two Cas1 dimers bridged by a Cas2 dimer.This heterohexameric complex binds a dual forked prespacer and catalyzes spacer integration using the terminal 3′-OH of each strand as an attacking nucleophile.Thus,the Cas1–Cas2 complex seems to play a dual role in both acquiring prespacer DNA and integrating it into the CRISPR array.CRISPR-Cas systems include two classes and could be subdivided into 6 types and more than 30 subtypes.Although the conserved Cas1 and Cas2 imply a common molecular mechanism for CRISPR-Cas adaptation,the fact that different factors involved in this stage in different CRISPR-Cas subtypes represents the intrinsic diversity of the CRISPR-Cas systems,such as Integration Host Factor(IHF)in Type IE,Cas9 and Csn2 in Type II-A,and Cas4 in type I,II,and V systems.As of yet,the high-resolution structural information of Cas1-Cas2 complex is limited to two species:Escherichia coli type I-E and Enterococcus faecalis type II-A CRISPR-Cas systems,adopted similar architectures.But there are differences in Cas1-Cas2 complex interface.Therefore,it remains to be investigated whether the structure of Cas1-Cas2 complex is diverse in different CRISPR-Cas systems.In this study,Pyrococcus furiosus(Pfu)Cas1-Cas2 was selected for crystallographic study to reveal the structural architectures of the Pfu Cas1-Cas2 complex.Analyze the interaction of Cas1-Cas2 in type I CRISPR-Cas system,and use the characteristics of the interaction of Cas1-Cas2 to assemble new adaptation modules,not only enrich the diversity of adaptation modules,but also provide more options for the development of CRISPR-Cas system as a biological recorder.The details are introduced as follows:Part 1: Structure of Pyrococcus furiosus Cas1–Cas2 complex highlights the diversity of CRISPR–Cas adaption module1.Pfu Cas1 and Cas2 form a stable complexThe P.furiosus genome encodes three subtypes of CRISPR–Cas systems which share one set of adaption module,including Cas1,Cas2 and Cas4.Pfu Cas1 and Cas2 could form a stable complex as measured by gel-filtration chromatography and pulldown assays.While the N-terminal MBP-tagged Cas2 binds to untagged Cas1,Nterminal MBP-tagged Cas1 could not bind to untagged Cas2,indicating the interaction between Cas1 and Cas2 involves the N-terminus of Cas1 but not that of Cas2.The direct interaction between Cas1 and Cas2 was further confirmed by isothermal titration calorimetry(ITC)and the KD of this interaction was determined to be ～4.38 μM.The calculated stoichiometry of Cas2 to Cas1 was ～0.35.2.Pfu Cas1–Cas2 complex has a distinct architectureWe have solved the crystal structure of Pfu Cas1-Cas2 complex,which is a heterohexameric complex including a Cas2 dimer in the middle and two Cas1 dimers bound to either side.Cas1 a and Cas1 c contact with the Cas2 dimer,while no contact between Cas1 b or Cas1 d and the Cas2 dimer has been observed.The Cas2 homodimer surface where the signature catalytic D9 residue locates(named top surface)is exposed and available for DNA binding.However,the opposite surface(named bottom surface)is almost buried by the two Cas1 dimers.Although all adopting a butterfly shape,the architecture of Pfu Cas1-Cas2 complex is distinct from that of E.coli or E.faecalis: the wings(Cas1 dimers)of Pfu Cas1-Cas2 switch to a nearly opposite direction.Pfu Cas2 uses its C-terminal tail to interact with Cas1 N-terminal β1 in a parallel mode,which is similar to the interaction observed in E.faecalis Cas1-Cas2 complex.However,the Cterminal tail of E.faecalis Cas2 is longer than that of Pfu Cas2 and makes a turn,thus the parallelly bound Cas1 dimers of these two species switch to approximately opposite directions.Different lengths and the flexibility of the Cas2 C-termini raise the possibility of different binding modes for Cas1-Cas2 complexes.3.Mutagenesis study on Pfu Cas1-Cas2 complex interfaceThe interactions between Pfu Cas1 and Cas2 are mainly mediated by the Nterminus of Cas1 and the C-terminus of Cas2.To confirm that this observed interaction mode is physiological,we have performed mutagenesis studies on the residues located at the Cas1-Cas2 interface.Pull-down assays showed that deletion of the C-terminal 5residues of Cas2 or the β1 fragment of Cas1 completely eliminated the interaction.Alanine substitution of K4 within Cas1 N-terminal tail or E82 or E83 within Cas2 Cterminal tail weakens the Cas1-Cas2 interaction,and the electricity inversion of the same residues significantly enhances the weakening effect.The mutants K4 E of Cas1 and E83R of Cas2 resulted in a similar elimination as the truncations,indicating that these two residues play essential roles in the Cas1-Cas2 binding.We have observed two hydrogen bonds outside Cas1 N-terminus and Cas2 C-terminus: Y22 and T282 of Cas1 and R66 and N56 of Cas2,respectively.Alanine substitution on these residues does not make any effect Cas1-Cas2 binding,indicating these interactions may be dispensable for the Cas1-Cas2 interaction.EMSA further analyzed the effects of these mutations on the complex formation of Cas1-Cas2.Here we used the prespacer DNA27-bp duplex with 5-nt 3’ overhangs to evaluate the DNA binding ability of the wild type and mutant Pfu Cas1 or/and Cas2.Pfu Cas1 alone could bind the prespacer DNA.Although Cas2 alone could not bind DNA,it significantly enhances the DNA binding ability when forming a complex with Cas1.EMSA further confirmed the disruption of the association of Cas1 and Cas2 by these mutations.4.Mapping of DNA binding region of Pfu Cas1-Cas2 complexThe complex formed between Cas1-Cas2 and prespacer depends on the interaction of charge.To identify the prespacer binding regions on Pfu Cas1-Cas2 complex,we analyzed of the electrostatic surface potential of Pfu Cas1-Cas2 by Py Mol.The top or bottom surfaces of Cas2 dimer are positively charged.We carried out mutagenesis studies to reverse the electricity of the positive-charged spots on the top surface(K16,K18,K19,and R22)and the bottom surface(K64 and R66)of Cas2 dimer,and evaluated the DNA binding ability of these mutants by EMSA(Fig.4a and4b).The EMSA results indicate that the Cas2 dimer top surface,but not the bottom surface,is responsible for the prespacer binding.Mutations of two positive-charged spots close to the Cas1 homodimer interface(H45-H54-H68-R79 and R192-K269-K273)significantly decreased the DNA binding ability,indicating that this region is on the pathway of 3’ overhangs threading through.By structural comparison,the catalytic residues of Pfu Cas1 are E149,H214,and E229.There are two positive-charged spots located close to this catalytic site.Mutant of the upside spot(K98-R101-K141)decreased the DNA binding ability,while the downside spot(R153-R173-K232)did not.The spot(R242-K246-K281)close to the Cas2 dimer moderately decreased the DNA binding ability.Pull-down assays show that these mutations have no effect on the Cas1-Cas2 complex formation.5.The prespacer binding model for Pfu Cas1-Cas2 complexThe two monomers within Cas1 asymmetric dimer adopt different conformations and are likely to have different biological functions.Within the Cas1-Cas2 complex,one subunit contact with Cas2,and the distal noncontacting Cas1 is the catalytic subunit.Compared to the Cas1-Cas2 complex structures of E.coli and E.faecalis,the distinct architecture of Pfu Cas1-Cas2 complex makes the distal Cas1 locating further away from the Cas2 dimer,indicating that the prespacer needs a much longer 3’ ss DNA overhang to reach the catalytic site of the distal Cas1.Based on the reported Cas1-Cas2-prespacer models and our structural and mutagenesis results,here we proposed a prespacer binding model for Pfu Cas1-Cas2 complex.We model a prespacer containing a mid-duplex of 15-bp and two 11-nt 3’ overhangs.The duplex DNA binds to the top surface of Cas2 homodimer,and the 3’ overhangs flip away from the duplex and thread through the C-terminal domain of the distal Cas1 and end at the Cas1 catalytic site.We compared the binding affinity of different prespacers.The EMSA showed that Pfu Cas1-Cas2 complex preferred the 37-bp prespacer with 5-nt 3′ overhangs to that with11-nt 3′ overhangs,and a shorter splayed prespacer with a 15-bp mid-duplex and 5-nt3′ overhangs showed a much weaker binding.Part Ⅱ Engineering of CRISPR-Cas adaption module1.Analysis of Cas1-Cas2 interaction of the type I CRISPR-Cas system adaptation modulesCas1 and Cas2 are universally conserved across all CRISPR-Cas subtypes.The available Cas1-Cas2 complex structures from two species,E.coli and E.faecalis,adopted similar architectures.This study explores whether the architectures of Cas1-Cas2 are conserved in type I CRISPR-Cas system.Through pull-down assays of the interaction of Cas1 and Cas2 in type I CRISPRCas system subtype I-A Archaeoglobus fulgidus,I-B Clostridium kluyveri,I-C Bacillus halodurans,I-D Synechocystis sp.PCC 6803,we found that MBP-Cas1 cannot be combined with Cas2,while MBP-Cas2 can be combined with Cas1.Consistent with the pull-down results of Pfu Cas1 and Cas2,it is implied that the N-terminus of Cas1 may be involved in the interaction between Cas1 and Cas2.Based on the characterization of protein structure comparison,the N-terminal β1 of Cas1 and the Cterminal 5 contiguous residues of Cas2 were deleted respectively,disruption of the association of Cas1 and Cas2.The above results suggest that the combination of Cas1 and Cas2 complexes of the Type I CRISPR-Cas system are highly conserved,and mainly depend on the interaction between the N-terminal β1 of Cas1 and the C-terminal tail of Cas2.2.Assemble new adaptation modules via natural Cas1-Cas2Using the characterization of the conservative combination of Cas1-Cas2,the Type I CRISPR-Cas system adaptation module,the new adaptation modules are assembled.The results of pull-down assays suggest that I-A Cas1 have a weaker interaction with the above species MBP-Cas2,I-C Cas1 is not bound with the above species MBP-Cas2,I-D Cas1 is combined with I-C MBP-Cas2,and Pfu Cas1 has a very strong combination with the above species MBP-Cas2.Furthermore,the combination of the newly assembled Cas1-Cas2 complex still relies on the N-terminalβ1 of Cas1 and the C-terminal tail of Cas2.Removing N-terminal β1 of I-A and Pfu Cas1,or removing the C-terminal tail of the MBP-Cas2,can disrupt the assembly of the new adaptation module.3.Assemble new adaptation modules via modifying Cas geneThe Cas1-Cas2 of the I CRISPR-Cas system is conservatively interacted and can the new adaptation modules be assembled by retrofitting the Cas1 and Cas2 structures?First,it was explored whether the replacement of the N-terminal β1 of Cas1 could be interacted between Cas1 and Cas2,which could not be interacted.I-D Cas1 and Pfu Cas2 cannot be interacted to replace I-D Cas1 β1 as Pfu Cas1 β1,basing on the structure of I-D Cas1 and Pfu Cas1.The results of pull-down assays suggest that the modified ID Cas1β1 did not bind to Pfu Cas2.Further inquiry is explored whether the replacement the C-terminal tail of the MBP-Cas2 could be interacted between Cas1 and Cas2.Previous experiments have shown that I-C Cas1 and I-A,I-B,I-D and Pfu Cas2 cannot form Cas1-Cas2 complex.Replace the C-terminal tail of the I-A,I-B,I-D and Pfu Cas2β5 chains with the C-terminal tail of the I-C Cas2 respectively,and the Pull-down analysis found that the modified Cas2 was able to interact with Cas1.In addition,previous experiments have shown that I-D Cas1 and I-A,I-B,I-D and Pfu Cas2 cannot form Cas1-Cas2 complex.Replace the C-terminal tail of the I-A,I-B,and Pfu Cas2 β5 chains with the C-terminal tail of the I-D Cas2 respectively,and the Pull-down analysis found that the modified Cas2 was able to interact with Cas1 as well.The above experiments suggest that the new adaptation module can be assembled by modifying the C-terminal tail of cas2.In summary,we have solved the crystal structure of Pfu Cas1-Cas2 complex by crystallographic study,which has a distinct architecture from E.coli and E.faecalis.The direction of the two Cas1 dimers is driven by Cas2 C-terminal tail in the Pfu Cas1-Cas2 complex,which results in two properties.One is Pfu Cas1-Cas2 complex having a distinct architecture,the other is a different prespacer binding mode.Basing on our structural and mutagenesis results,we modeled a prespacer with shorter duplex and longer 3′ overhangs to bind Pfu Cas1–Cas2 complex.Our study highlights the diversity in the CRISPR adaptation modules.The interaction of Cas1 and Cas2 in type I CRISPR-Cas system is highly conserved,and mainly depends on the interaction between the N-terminal β1 of Cas1 and the C-terminal tail of Cas2.Using the characterization of the conservative combination of Cas1-Cas2,the new adaptation modules are assembled via natural or modifying Cas gene.The rich composition of Cas1-Cas2 adaptation modules provides more possibilities for the development of the CRISPR-Cas system Cas1-Cas2 as a biological recorder. |
