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Structure of N-Terminal Domain of ZAP Indicates How a Zinc-Finger Protein Recognizes Complex RNA

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Structure of N-Terminal Domain of ZAP Indicates How a Zinc-Finger Protein Recognizes Complex RNA ARTICLES Structure of N-terminal domain of ZAP indicates how a zinc-finger protein recognizes complex RNA Shoudeng Chen1,2,4, Yihui Xu3,4, Kuo Zhang1,2, Xinlu Wang3, Jian Sun1,2, Guangxia Gao3 & Yingfang Liu1 Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, such as HIV-1, by targeting viral mRNA for degradation. How ZAP recognizes its target RNA has been unclear. Here we report the crystal structure of the N-terminal domain of rat ZAP (NZAP225), the major functional domain. The overall structure of NZAP225 resembles a tractor, with four zinc-finger motifs located at the bottom. Structural and functional analyses identified multiple positively charged residues and two putative RNA-binding cavities forming a large putative RNA-binding cleft. ZAP molecules interact to form a dimer that binds to a ZAP-responsive RNA molecule containing two ZAP-binding modules. These results provide insights into how ZAP binds specifically to complex target RNA. ZAP is a host factor that specifically inhibits the replication of certain organized to form a previously undescribed structure but also viruses, including HIV-1 (ref. 1), Ebola virus2 and Sindbis virus3. provide insights into how ZAP functions. ZAP does not induce a universal antiviral state, as some viruses grow normally in ZAP-expressing cells3. ZAP binds directly to specific viral RESULTS mRNA sequences4, recruits cellular poly(A) RNase (PARN) to shorten Overall structure of NZAP225 the poly(A) tail and recruits a 3′–5′ exoRNase complex, the RNA exo- Bacterially expressed NZAP225 was purified to homogeneity. Crystals some, to degrade target mRNAs from the 3′ end1. ZAP also recruits the diffracting to a resolution of 1.8 Å were obtained, and the structure of decapping complex through its cofactor RNA helicase p72 to initi- NZAP225 was determined by single-wavelength anomalous disper- ate 5′–3′ degradation of target mRNAs1,5,6. No obvious common ele- sion using the bound zinc atoms within the protein. The structure was ments or motifs have been identified in ZAP target RNAs; their only refined to a final Rwork 19.1% and Rfree 22.8%. The final experimental common feature is that they are more than 500 nucleotides long. electron density map shows that all residues except residues 55–60 © 2012 Nature America, Inc. All rights reserved. America, Inc. © 2012 Nature There are four tandem CCCH-type zinc-finger motifs in the were traced well (Fig. 1a). Data collection, phasing and refinement N-terminal domain of ZAP7. A fusion of the N-terminal 254 amino statistics are presented in Table 1. acids of ZAP with the gene product that confers zeocin resistance The structure of NZAP225 consists of seven α-helices, three npg (NZAP-Zeo) has antiviral activity comparable to that of the full- β-strands, three short 310α-helices and several loops. This structure length protein7. This suggests that the N-terminal domain is the major resembles a tractor that can be divided into three layers: a ‘cockpit’ functional domain that binds target RNA and recruits the mRNA layer on top, ‘wheel guards’ in the middle and four ‘wheels’ at the degradation machinery. ZAP has recently been reported to interact bottom. The top cockpit layer consists of the first 65 residues and has with retinoic acid–inducible gene-I (RIG-I) and thereby enhance an αβ architecture with a twisted plane formed by three short antipar- RIG-I–mediated interferon production by facilitating RIG-I oligo- allel β-strands surrounded on top by three α-helices (Fig. 1b). The merization8. Notably, the RIG-I interacting region was also mapped four predicted zinc-finger motifs (ZF) are located at the four corners to the N-terminal domain of ZAP. of a twisted rectangle in the bottom layer (Fig. 1b). Each zinc finger The features of ZAP-responsive RNAs and the mechanism coordinates one zinc ion at the center via three cysteines and one by which ZAP specifically recognizes these RNAs have been histidine (Fig. 1b). The overall folds of the zinc binding domains and elusive. Furthermore, considering the relatively small size of the their sequences are different from each other (Fig. 1c). The top cock- N-terminal domain of ZAP, how ZAP binds a complex target pit layer and the bottom zinc-finger motifs are linked by four helices, RNA and recruits multiple components of the RNA degradation which form the middle layer. Each of these four helices interacts with machinery has been puzzling. To address these questions, we one zinc-finger motif: helix 4 with ZF1, helix 5 with ZF2, helix 6 determined the crystal structure of the N-terminal domain of rat with ZF3 and helix 7 with ZF4, and it is likely that the interactions (Rattus norvegicus) ZAP covering residues 1–225 (termed NZAP225). strengthen the loose structure of the zinc-finger motifs. A detailed Our results not only show how the four zinc-finger motifs are description of the interactions between the cockpit, wheel guards 1State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. 2Graduate School of the Chinese Academy of Sciences, Beijing, China. 3Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. 4These authors contributed equally to this work. Correspondence should be addressed to Y.L. ([email protected]) or G.G. ([email protected]). Received 14 November 2011; accepted 3 January 2012; published online 11 March 2012; doi:10.1038/nsmb.2243 430 VOLUME 19 NUMBER 4 APRIL 2012 NATURE STRUCTURAL & MOLECULAR BIOLOGY ARTICLES Figure 1 Overall structure of NZAP225. a b N (a) Ribbon diagram of NZAP225 structure. The �2 �3 cysteine and histidine side chains coordinated �2 to zinc atoms (orange) are shown in a ball-and- β3 Cockpit �1 �3 stick representation. The α-helices and β-sheets �1 β3 β2 β2 are numbered, and the character ‘η’ represents β1 β1 the 310-helix. (b) Topology diagram of the NZAP225 structure. (c) Tertiary structures and �5 Wheel sequence alignment of the zinc-finger motifs. �7 guards �4 �5 �6 �7 c �4 (d) Surface electrostatic distribution generated ZF4 η1 by PyMOL. Positively charged residues are in C H C η1 C C η3 C H C blue, negatively charged residues in red and �6 η3 Wheels η2 Zn Zn η2 Zn Zn neutral residues in white. C C ZF1 C C H C H C ZF2 ZF1 ZF2 ZF3 ZF4 ZF3 infected with a VSVG-pseudotyped MLV-luc vector or transfected with pGL3-Luc-Na, a c Cys96 reporter containing a ZAP-sensitive fragment Cys162 4 Cys174 derived from the Sindbis virus (SINV) , and Cys78 Cys73 Cys182 His110 Cys168 antiviral activity was recorded in terms of fold inhibition. Although the proteins showed His86 Cys108 His191 Cys88 Cys150 different antiviral activities against the two Cys82 His172 Cys187 reporters, presumably resulting from the dif- ZF1 ZF2 ZF3 ZF4 ferent experimental systems used, the relative activities of the mutant proteins compared ZF1 ZF2 with wild-type ZAP were similar for both ZF3 reporters. Double- or triple-residue mutants ZF4 in the first, second and fourth zinc fingers (R74A R75A K76A, K89A R95A K107A and d R179A R189A, respectively) showed consid- erably reduced activity (Fig. 2c). In contrast, 90° 90° the antiviral activity of the triple mutant in the third zinc finger (R158A K159A Q160A) was only minimally reduced (Fig. 2c). The activities of single-residue mutants were either only marginally (R75A and K151A) Bottom view Side view Top view or moderately affected (R74A, K76A, R103A and R170A) (Fig. 2c). © 2012 Nature America, Inc. All rights reserved. America, Inc. © 2012 Nature and zinc-finger motifs can be found in Supplementary Figure 1. The ability of mutant proteins to bind target RNAs was analyzed These results suggest that this structure is rigid and is not likely to with an in vitro RNA binding assay4. SINV-derived RNA fragment Na undergo significant conformational change. Dali search results reveal was used for the binding assay, and RNA fragment Di, which fails to npg that the overall folding of NZAP is unlike any that have previously bind to ZAP, was used as a negative control for nonspecific binding. been described (data not shown). The specific RNA-binding ability of the multi-residue mutants (K89A R95A K107A and R179A R189A) was considerably reduced, whereas Roles of positively charged residues in the zinc-finger regions that of single-residue mutants (R74A, K76A, R103A and R170A) was On the bottom surface of the structure formed by the four zinc- little affected (Fig. 2d,e). Reductions in the in vivo antiviral activity finger motifs, 78% (22 of 28) of the basic residues are conserved of the mutants were more dramatic than those in in vitro RNA bind- (Supplementary Fig. 2). Some of these residues contribute to the ing activity, suggesting that, although these mutations did not have formation of a positively charged groove located between the front a major effect on ZAP RNA binding activity in our in vitro system, and the rear wheels (Fig. 1d). In ZF1, residues Arg74, Arg75 and they did strongly influence in vivo antiviral activity. This discrepancy Lys76 are located in the loop region between its first two cysteine between the antiviral activities and RNA-binding abilities of R74A residues. In ZF2, residues Lys89, Arg95, Arg103 and Lys107 extend and K76A may be accounted for by the possibility that the loss of these out from the bottom surface. Residue Arg103 clearly forms a bulge at positively charged residues is compensated for by adjacent positively a corner of the rectangle formed by the tractor wheels. In ZF3, similar charged residues.
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