Structural basis of viral RNA-dependent RNA PNAS PLUS polymerase catalysis and translocation

Bo Shua,b and Peng Gonga,1

aKey Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, ; and bUniversity of Chinese Academy of Sciences, 100049, China

Edited by Thomas A. Steitz, Howard Hughes Medical Institute, Yale University, New Haven, CT, and approved May 19, 2016 (received for review February 15, 2016) Viral RNA-dependent RNA polymerases (RdRPs) play essential roles NAC model featuring six reference states (10). The model starts with in viral genome replication and transcription. We previously reported astate1(S1) complex with a vacant active site that is in the catalyt- several structural states of the RdRP nucleotide addition ically open conformation and upon NTP binding proceeds to a state 2 cycle (NAC) that revealed a unique palm domain-based active site (S2) complex with the active site still in the open conformation. An closure mechanism and proposed a six-state NAC model including a important conformational change then takes place to position key hypothetical state representing translocation intermediates. Using catalytic residues and two magnesium ions around the priming nu- the RdRP from another human , enterovirus 71, here cleotide and the substrate NTP to achieve proper geometry of a we report seven RdRP elongation complex structures derived from closed active site for catalysis, yielding state 3 (S3) immediately before a crystal lattice that allows three NAC events. These structures and state 4 (S4) immediately after the phosphoryl transfer reaction. suggested a key order of events in initial NTP binding and NTP- As the catalytic geometry starts to disintegrate, the structural changes induced active site closure and revealed a bona fide translocation in the palm domain result in state 5 (S5) with an open conformation – intermediate featuring asymmetric movement of the template active site. State 6 (S6) is then a hypothetical translocation in- product duplex. Our work provides essential missing links in under- termediate state that bridges the pretranslocation S5 and the post- standing NTP recognition and translocation mechanisms in viral RdRPs translocation S1 in the next NAC. and emphasizes the uniqueness of the viral RdRPs compared with The six-state model has provided a framework for understanding other processive polymerases. the molecular details and the unique features of the viral RdRP elongation NAC. In the active site closure step, the notable RNA-dependent RNA polymerase | nucleotide addition cycle | translocation backbone conformational changes are limited to motifs A and D intermediate | enterovirus 71 | crystal structure in the palm domain. These conformational changes are in drastic contrast to those taken by the well-characterized A-family poly- – n recent years, several notable emerging infectious diseases have merases that use a large-scale rotational movement of the O-helix Ibeen caused by RNA , including highly pathogenic avian containing fingers domain to achieve the same closure step (11, 12). influenza viruses, Ebola , and Middle East respiratory syn- One implication behind this apparent difference in the active site drome coronavirus. RNA viruses are quite diverse in virus particle closure mode is that the mechanisms by which the polymerase se- BIOCHEMISTRY and genome structure and in virus entry and assembly mechanisms. lects the correct NTP substrate and induces active site reorganiza- However, they do share fundamental features in their genome tion for catalysis are also quite different. In A-family polymerases, replication and transcription, using a virally encoded RNA- the translocation step is coupled to the postcatalysis reopen- ing of the active site when a conserved tyrosine residue in the dependent RNA polymerase (RdRP) to carry out the biosynthesis of “ ” an RNA product directed by an RNA template. Although the ge- O-helix pushes the nascent base pair upstream in a motion that is the reverse of that observed during the active site closure nome replication machinery often requires the participation of other (11, 13). However, no intermediate structure between the pre- factors, typically at the initiation phase of synthesis, the RdRP governs the elongation phase of synthesis that includes thousands of efficient nucleotide addition cycles (NACs). Viral RdRPs vary greatly in size Significance and structural organization, from the ∼50-kDa 3Dpol (1, 2), to the ∼100-kDa flavivirus NS5 that contains a naturally fused RNA viruses encode a unique class of RNA-dependent RNA methyltransferase domain (3), to the ∼250-kDa nonsegmented neg- polymerases (RdRPs) to carry out their fully RNA-based genome ative-strand RNA virus L protein harboring at least three enzyme replication and transcription. Although the chemical nature of modules (4) and the ∼260-kDa three-subunit PA-PB1-PB2 influenza nucleotide addition is essentially shared by all nucleic acid poly- virus replicase complex (5). On the other hand, all RdRPs share a 50- merases, the structural and mechanistic details taken by each to 70-kDa polymerase core that forms a unique encircled right-hand polymerase class differ to various extents. Here we report seven structure with palm, fingers, and thumb domains. Among the seven crystal structures of enterovirus 71 RdRP elongation complex at – classic RdRP catalytic motifs, A–E are within the most conserved 2.5 2.8 Å resolution. In these structures the polymerases are palm domain, and F and G are located in the fingers; they are all poised at various distinct stages to reveal mechanistic details of arranged similarly around the active site (6–9). The structural con- initial NTP binding, key amino acid side-chain conformational servation of the RdRP polymerase core and the seven motifs form the switches during active site closure, and in particular the post- catalysis movement of the RNA duplex on the way to vacate the basis for understanding the common features in viral RdRP catalytic active site for the next nucleotide addition cycle. mechanism and for finding intervention strategies targeting these

enzymes with possible broad-spectrum potential. Author contributions: B.S. and P.G. designed research; B.S. performed research; B.S. and As with other classes of nucleic acid polymerases, the viral RdRP P.G. analyzed data; and B.S. and P.G. wrote the paper. elongation NAC comprises sequential steps of initial NTP binding, The authors declare no conflict of interest. active site closure, catalysis, and translocation. In a recent study using This article is a PNAS Direct Submission. CTP and deoxy-CTP analogs in the poliovirus (PV) RdRP elongation Data deposition: Crystallography, atomic coordinates, and structure factors reported in complex (EC) crystal-soaking experiments, the polymerase having a this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes guanine base at the +1 template position was successfully trapped at 5F8G–5F8J, 5F8L, 5F8M, and 5F8N). different stages of a single NAC, leading to the proposal of a working 1To whom correspondence should be addressed. Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1602591113 PNAS | Published online June 23, 2016 | E4005–E4014 Downloaded by guest on September 29, 2021 and posttranslocation states to refine the translocation process otides (i.e., CCU) at most, indicating a threshold for the crystal lat- further has been captured in A-family polymerases by crystallogra- tice to accommodate growth of the template–product duplex. We phy. Without an O-helix counterpart, viral RdRPs likely have then explored using this crystal lattice as a platform to capturing established unique conserved components to control translocation, important EC states within three consecutive NACs. By combining and their ECs may experience metastable intermediates that provide an incubation time scanning strategy (15, 16) and the use of different valuable details for identifying translocation-related protein com- NTP combinations in the native EC-soaking trials, we obtained seven ponents and the details of RNA movement during translocation. representative EC structures distributed in the first and the third In this study, we obtained a crystal form of the enterovirus 71 NACs (Fig. 1B and Tables 1 and 2). The resulting crystal lattices (EV71) RdRP EC that allows multiple nucleotide incorporations were nearly isomorphous based on unit cell dimensions and inter- in NTP-soaking trials. By using natural NTP substrate combina- complex packing modes (Tables 1 and 2 and Fig. 1C). This obser- tions and controlling the incubation time, we were able to capture vation is drastically different from the output of the soaking EC species that provide previously unidentified mechanistic details experiments using a PV RdRP EC crystal that also allows multiple for initial NTP binding, active site closure, and, in particular the nucleotide incorporation (14); in that lattice (named “PV_r5”)every RNA motion during translocation that shows an asymmetric translocation event resulted in obvious translational movement be- movement of the two strands in the template–product duplex. tween RdRPs within the aforementioned EC dimer as the growing RNA duplexes collided with each other. Results A Unique RdRP EC Lattice That Is Capable of in Situ Elongation for A Plausible Order of Events During Initial NTP Binding and Active Site Multiple NACs. Recently we developed an RNA-mediated crys- Closure. The isomorphous feature of the EV71 RdRP EC lattice tallization strategy that was highly effective in crystallizing and the fact that the EC can enter the third NAC mean the three picornaviral RdRP ECs (14). By providing a GC or GU sequence consecutive NACs occurred in an environment free of constraints overhanging the upstream end of the template–product duplex to brought by lattice variation, providing a valid platform for time- facilitate inter-EC contacts, an EC dimer typically becomes the resolved NTP-soaking experiments. We tested CTP soaking with minimal crystallizing unit, with the two upstream RNA duplexes various incubation times and obtained three representative states in interacting in the middle and two polymerases facing away from each the first NAC (denoted “C1” for “cycle 1”; Table 1). In the prior PV other. These RNA–RNA interactions play important roles in crys- RdRP EC work, a 2′,3′-dideoxy CTP (ddCTP)-derived structure tallization but also impose steric constraints for in-crystal soaking showed clear density for the entire ddCTP molecule, whereas the experiments designed to go through multiple NACs. By attempting RdRP conformation remained essentially unchanged around the to crystallize EV71 RdRP EC using a combination of RdRP from active site. This structure was assigned as the reference state 2 (S2) different viral genotypes and RNA with different lengths of tem- to represent a fully open conformation active site with a bound plate–product duplex, we have obtained a picornaviral RdRP EC NTP. However, such a state was not observed in all of the CTP- crystal form within which ECs are no longer organized as dimers. soaked EV71 RdRP EC C1 structures. If the active site confor- Instead, the upstream duplex points toward a spacious solvent mation remained fully open, only medium-level density for the CTP channel (Fig. 1A) that may allow multiple incorporation and trans- base moiety and weak-level density of the ribose was evident (C1S1/2 location events to occur in NTP-soaking experiments. structure, CTP not modeled; Fig. 2 A and B). This observation – The EC in this crystal was obtained after incorporating a (GA)3 indicates that the template NTP substrate base-pairing appears to hexa-nucleotide sequence into an RdRP–RNA binary complex be sampled before the rearrangement of NTP ribose, triphosphate, containing an 8-bp template–primer duplex. The remaining and the surrounding active site motifs toward the in-line catalytic “GGACCU ...” template sequence was designed to direct sub- geometry. Defined density next to residue D330 (the second aspartate sequent in-crystal elongation, as may be allowed by this particular in the RdRP hallmark motif C sequence XGDD) with an interacting lattice (Fig. 1B). When CTP, UTP, and GTP are provided, the EC is distance of 2.1 Å to a side-chain oxygen indicated a bound magne- expected to incorporate five nucleotides (i.e., CCUGG). However, sium ion (“metal A” or MeA according to consensus nomenclature) even with overnight incubations, the EC incorporated three nucle- that is required for subsequent catalysis.

Fig. 1. A unique EV71 RdRP EC crystal lattice allows multiple nucleotide-incorporation events. (A)2Fo-Fc electron density map (contoured at 1.5 σ with a radius of 30 Å, in cyan) around the upstream end of one EC (polymerase in green, RNA strands in red and blue) shows a spacious channel to accommodate the growth of RNA duplex. The black dot indicates the center of the map, and symmetry-related neighboring ECs are labeled as Sym1/2/3. (B) RNA sequence flanking the active site

of the native EC (C1S1) and those of the other six complexes derived from the native EC in crystal-soaking trials. The template is in cyan, and product is in green. The black box indicates the nucleotides incorporated during soaking. “C” and “S” in complex names stand for “cycle” and “state,” respectively, and the subscript

numbers reflect the assigned cycle/state numbers. (C) The isomorphous nature of the EC lattices. C1S1/2 and C3S6 structures (bold-faced in B) were chosen as representatives to indicate the very limited lattice alteration upon three rounds of nucleotide incorporation. The superimposed polymerases (using the

traditional least-squares method) are on the right side with the C1S1/2 complex taking a coloring scheme indicated by individual parts of the EC and the C3S6 complex in black; their symmetry-related neighboring ECs are colored in orange (C1S1/2) and purple (C3S6).

E4006 | www.pnas.org/cgi/doi/10.1073/pnas.1602591113 Shu and Gong Downloaded by guest on September 29, 2021 Table 1. X-ray diffraction data collection and structure refinement statistics (set 1) PNAS PLUS NAC state* (PDB ID code)

C1S1 (5F8G) C1S1/2 (5F8H) C1S2/3 (5F8I) C1S4 (5F8J)

† Data collection

Space group P212121 P212121 P212121 P212121 Cell dimensions a, b, c,Å 63.1, 77.6, 153.6 63.3, 77.1, 149.9 63.7, 77.3, 149.7 63.4, 76.6, 149.3 α, β, γ, ° 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution, Ň 60.0–2.78 (2.88–2.78) 60.0–2.45 (2.54–2.45) 60.0–2.50 (2.59–2.50) 60.0–2.66 (2.76–2.66)

Rmerge 0.117 (0.50) 0.062 (0.51) 0.060 (0.48) 0.073 (0.50) I/σI 13.6 (3.2) 21.4 (3.3) 20.1 (2.9) 17.7 (2.5) Completeness, % 97.6 (98.4) 99.7 (99.9) 99.6 (100.0) 98.1 (96.4) Redundancy 5.4 (5.5) 4.7 (4.8) 4.3 (4.3) 4.9 (4.9) Refinement Resolution, Å 2.78 2.45 2.50 2.66 No. reflections 19,258 27,772 26,095 20,430 § Rwork/Rfree ,% 19.3/24.2 19.8/24.4 19.9/24.4 18.3/22.8 No. atoms Protein/RNA 3,681/471 3,681/471 3,681/471 3,685/491 Ligand/ion/water –/ 1/115 5/2/141 40/3/144 25/3/111 B-factors Protein/RNA 42.6/53.2 52.3/60.8 57.5/72.2 50.4/62.8 Ligand/ion/water –/18.6/38.9 36.3/44.4/49.8 53.0/53.2/55.5 43.6/39.1/48.1 RMSD Bond lengths, Å 0.008 0.008 0.008 0.008 Bond angles, ° 1.13 1.13 1.12 1.17 Ramachandran statistics¶ 91.2/8.3/0.2/0.2 93.1/6.6/0.0/0.2 92.9/6.9/0.0/0.2 92.6/6.9/0.2/0.2

*Coding scheme: C, cycle; S, state; subscript numbers reflect the cycle and state numbers: x/(x+1) indicates an NAC species between state x and the next

reference state. Soaking strategy: C1S1/2: CTP for 4 min; C1S2/3 – CTP for 4 min, and then transfer to UTP for 10 min; C1S4: CTP for 5 min 10 s. † One crystal was used for data collection for each structure. ‡Values in parentheses are for highest-resolution shell. § 5% of data are taken for the Rfree set, and the same Rfree set is applied for all structures. ¶Values are in percentage and are for most favored, additionally allowed, generously allowed, and disallowed regions in Ramachandran plots, respectively. BIOCHEMISTRY

If clear triphosphate density was visible, then key conformational moved 5.5 Å to be coordinated simultaneously by D233, D329, and changes around the CTP ribose 2′ and 3′ hydroxyl groups had al- D330 in the catalytically competent conformation (Fig. 2C). ready occurred, but the active site had not yet fully closed, leading to an intermediate state we call “S2/3” (C1S2/3 structure; Fig. 2A). The Postcatalysis Complexes in Two NACs Provide the NTP Selection conserved motif A residue D238 experienced a hallmark side-chain Details by +1 Template Purine Bases. Many DNA-dependent po- rotamer change to accommodate the ribose hydroxyls and estab- lymerase ECs contain a preinsertion site (the “E-site” where “E” lished a hydrogen-bonding network with the ribose and motif B stands for “entry”) in which the +1 template nucleotide is poised – residue S289 (Fig. 2C). As in the C1S1/2 structure, metal A still re- to allow initial NTP binding (13, 18 20). This site allows the sided several Ångstroms away from its catalytic position, whereas nascent base pair to form without establishing the stacking in- metal B was observed within coordination-distance range of the CTP teractions with the −1 base pair. To achieve active site closure, phosphates and the motif A residue D329. D329 (the first aspartate an NTP repositioning step needs to take place to move the NTP in sequence XGDD) is one of two universally conserved aspartic acid into the insertion site (the “A-site” where “A” stands for “addition”), residues in polymerases following the two-metal-ion catalytic mech- and this repositioning usually is accompanied by relatively large anism (17). However, this structure is not in the fully closed cata- conformational changes in the vicinity of the active site (11–13, 18). lytically competent state because the other universal aspartic acid These features in general permit the NTP selection process to occur residue located at position 233 in motif A is not in place. As char- at two distinct sites that have different sets of interactions, possibly acterized in the PV RdRP EC study, D233 features the most notable improving nucleotide selection fidelity. In contrast, in viral RdRP backbone movement during active site closure to achieve co- ECs the +1 template nucleotide is prestacked on the −1basepair, ordination with both metal ions. The capture of EV71 RdRP in a and as a result the initial NTP binding is to a site nearly identical partially closed state, S2/3, provides further evidence that the likely to the catalytic insertion site. Active site closure involves only order of events during active site closure is as originally suggested limited backbone shifts in motifs A and D with several hallmark by the PV study (10). In such a proposal, the precise placement of side-chain rotamer changes as mentioned above. The NTP selection ribose 2′ and 3′ hydroxyls triggers the reorganization around the by the RdRP EC therefore is structurally less complicated, because ribose, including residue D238, which in turn induces the move- the initial NTP binding and subsequent catalysis occurs in very similar ment of the D233 region within the same motif for metal ion co- protein environments. In addition to the C1S1 native structure and ordination and catalysis. C1S4 structure that show the details of CTP selection with a guanine A fully closed postcatalysis state in the EV71 structure was obtained base at the template +1 position (denoted “+1G:C”), we obtained + also (C1S4 structure; Fig. 2A) in which the details of the active site C3S1 and C3S4/5 structures showing UTP selection with a 1 adenine + were essentially identical to the S4 structures seen in the PV study. base ( 1A:U) (Fig. 3). When comparing the NTP-free S1 structures to Relative to their location in the C1S2/3 structure, the D233 side-chain the NMP-incorporated S4 and S4/5 structures by a maximum like- carboxylate group moved about 3.8 Å and rotated ∼120°, and metal A lihood superimposition of the polymerase molecules (21), the

Shu and Gong PNAS | Published online June 23, 2016 | E4007 Downloaded by guest on September 29, 2021 Table 2. X-ray diffraction data collection and structure refinement statistics (set 2) NAC state* (PDB ID code)

C3S1 (5F8L) C3S4/5 (5F8M) C3S6 (5F8N)

† Data collection

Space group P212121 P212121 P212121 Cell dimensions a, b, c,Å 62.3, 76.7, 151.2 63.6, 76.7, 150.1 63.7, 77.6, 151.4 α, β, γ, ° 90, 90, 90 90, 90, 90 90, 90, 90 Resolution, Ň 60.0–2.81 (2.91–2.81) 60.0–2.83 (2.93–2.83) 60.0–2.47 (2.56–2.47)

Rmerge 0.102 (0.50) 0.065 (0.50) 0.047 (0.51) I/σI 18.6 (4.0) 18.0 (2.6) 23.1 (2.6) Completeness, % 99.9 (100.0) 95.8 (96.1) 95.1 (94.8) Redundancy 6.4 (6.6) 3.7 (3.8) 4.1 (3.9) Refinement Resolution, Å 2.81 2.83 2.47 No. reflections 18,195 17,637 25,817 § Rwork/Rfree ,% 20.0/23.8 19.3/23.2 19.4/23.5 No. atoms Protein/RNA 3,677/468 3,681/465 3,677/360 Ligand/ion/water –/1/96 20/3/78 20/1/138 B-factors Protein/RNA 50.4/73.5 61.6/79.4 60.2/70.4 Ligand/ion/water –/46.9/45.2 56.3/49.2/49.8 54.5/42.2/58.5 RMSD Bond lengths, Å 0.009 0.009 0.008 Bond angles, ° 1.16 1.20 1.13 Ramachandran statistics¶ 91.4/8.3/0.0/0.2 91.7/7.8/0.2/0.2 91.7/7.8/0.2/0.2

*Coding scheme is the same as in Table 1. Soaking strategy: C3S1: CTP for 16 h; C3S4/5: CTP for 5 min, and then transfer to CTP/UTP/GTP for 20 min; C3S6: CTP/ UTP for 16 h. † One crystal was used for data collection for each structure. ‡Values in parentheses are for highest-resolution shell. § 5% of data are taken for the Rfree set, and the same Rfree set is applied for all structures. ¶Values are in percentage and are for most favored, additionally allowed, generously allowed, and disallowed regions in Ramachandran plots, respectively.

placement of the +1 template base was subjected only to very subtle serve as the general base in the proposed two-proton transfer movement toward the major groove side (Fig. 3A). The polymerase mechanism (22, 23). The water molecule coordinating metal B in active site conformation is essentially identical in the two product the C1S4 structure is about 3.0–3.1 Å away from two phosphate structures, with key residues D238, D233, S289, G290, and R174 oxygen atoms of the pyrophosphate, and a structurally equivalent following the same conformational switches for both NTPs. These water molecule has not been observed in RdRP structures. Along residues, together with motif F residue K159 sitting on the major with the previously proposed motif D lysine (K359 in PV RdRP) groove side and residue I176 that stacks onto the template base from (24) and motif F arginine (R174 in PV RdRP) (10) residues, this the downstream side, define a compact substrate pocket for the final water molecule also may be a candidate to protonate the β-phos- precatalysis fidelity checkpoint. The 2′ and 3′ hydroxyls of the na- phate of the substrate NTP as a general acid. scent NMP ribose were precisely placed toward D238, with the positional deviation of the both hydroxyl oxygen atoms in C1S4 A Previously Unidentified Translocation Intermediate Suggests an – and C3S4/5 complexes being within 0.2 Å. This observation sug- Asymmetric Movement of Template Product RNA Duplex. Translocation gests that the establishment of the interaction network around the has the net effect of changing the footprint of the polymerase NTP ribose hydroxyls is a key determinant for active site closure on nucleic acid by one nucleotide register and represents an and likely precedes and triggers the structural rearrangements important postcatalysis event that completes the NAC. Local, around the catalytic metal ions. if not major, conformational changes must take place to The two postcatalysis structures reported here are nearly iden- achieve translocation, and higher-energy intermediates may tical in polymerase active site conformation. However, only the exist between the pre- and posttranslocation states. However, C1S4 structure contains all 12 coordination partners for the two such translocation intermediates often are difficult to capture by + metal ions, with the 11 of these found within Mg2 coordination experimental biology because of their short-lived nature. Com- distances (Fig. 3B). Therefore, we consider C1S4 a bona fide pared with in-solution approaches, in-crystal polymerase synthesis postcatalysis S4 structure. In contrast, the coordination geometry may increase the possibility of capturing such intermediates by has begun to disintegrate in the C3S4/5 structure, and it therefore providing extra constraints through the packing environment of should be considered an intermediate between S4 and S5,evenif each polymerase. Indeed, the time scale for a single NAC in the the protein active site conformation remains closed. Note that current lattice is on the order of minutes (Tables 1 and 2), at least 2+ two water molecules participate in Mg coordination in the C1S4 100-fold slower than that measured in the solution studies in PV structure. The water molecule coordinating metal A is about 3.5 Å RdRP (25, 26). Ideally, the EV71 RdRP EC lattice reported here away from the 3′ hydroxyl oxygen. In the norovirus RdRP-RNA- could allow the capture of such intermediates in all three NACs. CTP crystal structure, a structurally equivalent water molecule To date, we have not successfully obtained any translocation in- + coordinates the metal A (a Mn2 in that case) and was suggested to termediate within the first or the second NAC, even after numerous

E4008 | www.pnas.org/cgi/doi/10.1073/pnas.1602591113 Shu and Gong Downloaded by guest on September 29, 2021 PNAS PLUS BIOCHEMISTRY

Fig. 2. CTP-derived sequential EC structures demonstrate a plausible order of precatalysis NAC events. (A) Four-cycle 1 (C1) NAC structures arranged in a sequential order with composite simulated-annealing (SA) omit electron density maps contoured at 1.2 σ.C1S1 represents the starting state 1 with a vacant active site; C1S1/2 shows evidence of initial NTP binding via base-paring; C1S2/3 shows the initial conformational change around D238 that is triggered by ribose 2+ hydroxyls during active site closure; C1S4 represents the bona fide catalytically closed state 4 with 11 of 12 Mg ion contacts intact. Coloring scheme: template in cyan (+1 nucleotide in orange), product in green, palm in gray (YGDD sequence in magenta), ring finger in yellow, and metal ions in cyan. (B) Electron

density around the NTP site in the C1S1/2 structure indicates that base-paring interactions and placement of the NTP ribose likely form before the correct alignment of triphosphate moiety. (C) The change in the interaction network around NTP ribose (ribose cluster) and catalytic metal sites (metal cluster) + demonstrates the order of events upon NTP binding and active site closure. Red spheres indicate water molecules coordinating with Mg2 .

rounds of attempts. However, with overnight incubation in the position −7, despite various extents of RNA conformational distortion presence of CTP and UTP, the EC could be paused at a clear occurring further upstream (14). The polymerase global conformation translocation-intermediate state in which all regions of the tem- of the C3S6 complex is highly consistent with the other six complexes plate–product RNA duplex except for the backbone region from presented in this study (Fig. 4A). The RNA regions upstream of po- position −3 to position +1 of the template strand had undergone sition −7 and downstream of position +2 are largely disordered in the A movement in the upstream direction (Fig. 4 ,C3S6). To the best of C3S6 structure (Fig. 4B). Although a portion of RNA backbone our knowledge, this is the first reported crystallography-derived poly- electron density of the disordered upstream RNA is somewhat merase EC intermediate that demonstrates the global motion of the traceable, defined intercomplex contacts are lacking at both ends of template–product duplex during translocation, and it is conceptually the RNA construct. Taken together, the consistent polymerase con- different from the crystal structure of yeast RNA polymerase II (Pol formation, the absence of defined crystal contacts at both ends of the II) EC in complex with α-amanitin, in which only the intermediate RNA constructs, and the ability of polymerase to absorb the confor- conformation of the downstream DNA is observed (27). Note that the mational distortion induced at the upstream end of its RNA collec- intermediate RNA conformation in our C3S6 structure mostly likely tively support the validity of the C3S6 structure. represents a naturally occurring state rather than an artifact induced In comparison with the C3S4/5 pretranslocation structure within the by crystal-packing constraints. In the previous work describing the same NAC, in the C3S6 structure the entire product strand moved in method used in crystallizing picornaviral polymerase EC, we dem- the upstream direction (Fig. 5). With all base-paring hydrogen bonds onstrated that the polymerase with consistent global conformation maintained between the two strands, the product strand phosphates maintains the native RNA conformation from the active site to about moved about a half-register on average, with some variation

Shu and Gong PNAS | Published online June 23, 2016 | E4009 Downloaded by guest on September 29, 2021 (Fig. 5 B and C). The product riboses and bases also moved in the concentrated in any one region. In contrast, polymerase interactions same direction but with larger variation. The upstream-most nucleo- with the template strand are clustered in two distinct regions around tide resolved in the structure is the −7 position, whose base almost the −5to−3and−2to+1 nucleotides, with the latter having the reached the −8 position of the pretranslocation structure, but the most extensive contacts. Motifs B, F, and G converge at the −2to+1 downstream-most +1 base has moved by only about one fifth of a region and create a sharp turn in the template strand (Fig. 5A)that register (Fig. 5 B and C). The template strand lagged behind, with its also is observed in DNA-dependent RNA polymerases (28, 29). As a backbone phosphates at positions −2to+1 remaining locked at their result, the +2 template base is fully unstacked from the upstream pretranslocational positions, whereas the −7to−4 phosphates moved nucleotides and is tucked into a small surface pocket created by the upstream by less than a half-register on average (Fig. 5). Globally index and ring fingers (Fig. 5A) (14). Motif G residues T114 and assessed, the template–product RNA duplex had undergone an S115 pack against the template strand +1/+2 backbone linkage and asymmetric movement with the product strand preceding the tem- likely serve as a control point for template strand movement during plate strand and the upstream region leading the downstream region. translocation (9). At the upstream end of this interaction cluster With the aim of understanding this apparent strand asymmetry there is an unusual and conserved backbone conformation for the in viral RdRP translocation, we compared the details of the ribose–phosphate linkage of the template strand −2 nucleotide (14). protein–RNA interactions from the active site to position −7in Together, these two interactions appear to lock the −2to+1se- all three C3 structures (Fig. 5C). Except for the interactions with quence in place, specifically preventing this region of the template the newly incorporated UMP in the C3S4/5 structure, the inter- from moving while the remainder of the product–template RNA actions are largely the same in the pre- and posttranslocation duplex undergoes translocation in an asymmetric fashion. To achieve states. Interactions between the product strand and the poly- the final posttranslocation state, i.e., S1 of the next NAC, the special merase are fairly evenly distributed from −7to−1 without being backbone conformations at the S6 template +1/+2 linkage and the

Fig. 3. Watson–Crick base-pair geometry as a major determinant of NTP selection. (A) Interaction details for NTP selection directed by +1 guanine (C1S1 and C1S4) and adenine (C3S1 and C3S4/5). All polymerase molecules were superimposed, and the structures are shown as individual panels with the S1 complexes on the left and S4/S4/5 complexes on the right. The coloring scheme is the same for each complex and is the same as in Fig. 2 with the product RNA in green, template RNA in cyan, and the +1 template nucleotide in orange. The ribose C4′ atoms of the +1 nucleotides are shown as spheres to aid the structural comparison with the alignment grids. Key interactions around the ribose cluster upon active site closure, and base-pairing hydrogen-bonding interactions at the +1 position are indicated by green and pink dashed lines, respectively. K159- and R174-related interactions are shown in purple. (B) A comparison of the metal cluster of the two product complexes. Mg2+–oxygen distances greater than 2.5 Å are shown in green text to highlight the disintegration of the ion- 2+ coordination interactions in the postcatalysis S4/5 complex. Red spheres indicate water molecules coordinating with Mg .

E4010 | www.pnas.org/cgi/doi/10.1073/pnas.1602591113 Shu and Gong Downloaded by guest on September 29, 2021 −2 position need to transition to the corresponding regions one base PNAS PLUS step downstream, and the energy to achieve the requisite confor- mational changes is likely an important contributor to the ultimate energy barrier for translocation. We next compared the pretranslocation C3S4/5 structure with the translocation-intermediate C3S6 structure. Although the interactions between the template strand and the polymerase remained largely unchanged, more than half of the residues par- ticipating in product strand contacts changed their interaction modes by interacting with a different site, losing original interactions or establishing additional interactions (Fig. 5C). This finding is consis- tent with the observation that the interactions between the poly- merase and the product RNA are relatively nonintensive and evenly distributed. We therefore propose that the product RNA strand may be capable of sliding back and forth between its pre- and post- translocation states. In contrast, the movement of the template RNA is controlled more stringently by the polymerase, in particular around the −2to+2 region. The backbone conformational changes required to translocate the +1/+2 junction and the −2ribose–phosphate linkage are likely accompanied or assisted by local polymerase con- formational changes that also require energy, making the final step in translocation rate-limiting and largely irreversible (Fig. 5C). Discussion An Improved View of Viral RdRP Elongation NAC. Using the advan- tages provided by a particular EV71 RdRP EC lattice, the cur- rent study makes important advances toward the understanding of the viral RdRP elongation NAC (Fig. 6). The current NAC working model starts with an S1 complex in the absence of NTP substrate with an open active site; after diffusing into the NTP entry Fig. 4. The C3S6 complex is a translocation intermediate, has a global poly- channel formed between the motif F in the ring finger and motifs merase conformation consistent with other complexes, and is free of defined A and D in the palm, the NTP establishes its initial interaction intercomplex interactions at either end of its RNA constructs. (A) Stereo-pair through base-paring with the +1 template base and stacking with the images of all seven complexes with their polymerases superimposed. The C3S6 −1 priming base (S ), leading to the rearrangement around the NTP complex is shown in black, and the other six complexes are colored as the C1S1/2 1/2 complex in Fig. 1C.The+2 template nucleotides in all complexes are omitted for ribose and motif A residue D238 and finally bringing the NTP tri- clarity. The −1and−8 position labels correspond to the regular translocational BIOCHEMISTRY phosphate, two divalent metal ions, and another motif A residue, positions defined by the six nontranslocation-intermediate complexes. (B, Upper)

D233, into place to achieve the catalytically closed conformation (S3) The complete sequence of the RNA constructs in the C3S6 complex is shown with as observed in a norovirus polymerase–RNA–CTP complex structure gray font indicating unresolved nucleotides in the structure. (Lower Left)A (23). Immediately after the phosphoryl transfer reaction (S4), the portion of the unresolved upstream RNA backbone in the C3S6 structure is catalytic geometry around the metal ionsstartstodisintegrate,andthe somewhat traceable. (Lower Right) The downstream RNA is essentially disor- dered. Electron densities for defined crystal contacts are lacking at both ends of active site reopens (S5); translocation begins with the less-restrained product RNA and the upstream region of the template–product du- the RNA construct. Cyan mesh indicates the 2Fo-Fc electron density map con- toured at 1 σ; green/red mesh indicates the Fo-Fc electron density map contoured plex (S6) and then finally needs to overcome the conformational − + at 3 σ. Green ribbons indicate polymerase; red/blue ribbons indicate template/ transition around the 2to 2 region of the template strand to be- product RNA. Black dots indicate the center of the map. The map radius is 30 Å. come the posttranslocation S1 complex of the next NAC (Fig. 6A).

On NTP Selection and Fidelity Control by Viral RdRP. Viral RdRPs typ- nascent NMP ribose hydroxyl groupsinthetwoproductcomplexes −4– −5 – ically have misincorporation rates in the range of 10 10 (30 33) emphasizes the importance of the ribose–Aspswitchinsubstratese- and therefore ought to be considered as medium-fidelity polymer- lection. We propose that the RdRP specific ribose–Aspswitchisa ases compared with high- and low-fidelity representatives (26, 34, positive contributor to the overall fidelity for viral RdRPs and to some 35). Therefore, the NTP selection by viral RdRP, in particular for its extent may compensate for the loss of one fidelity checkpoint resulting EC, is expected to be reasonably stringent. Because a preinsertion from the lack of a preinsertion site in these polymerases. site is missing in viral RdRP ECs, the NTP selection occurs in a limited space in an induced-fit manner with only local conforma- On the Translocation Mechanism by Polymerases. Two major theories tional changes. It has been proposed that the precise recognition of have been proposed to describe the central mechanism of poly- the equivalent geometry of the Watson–Crick base pairs may be merase translocation, namely the Brownian ratchet model and the the most important factor in NTP selection by polymerases (36). power stroke model (11, 37–39). The Brownian ratchet model Although we have not obtained structural data for all four RNA base pairs at the RdRP +1 position, the +1G:C and +1A:U product features a fast equilibrium between the pre- and posttranslocation states, and it allows possible intermediates in the absence of the next complexes (C1S4 and C3S4/5) strongly support this proposal, because the spatial placement of the +1 nucleotides, the edges of the +1base incoming NTP, but the presence of a bound NTP strongly stabilizes pair, and the shape of the active site complementing the +1 nucleotides the polymerase in the posttranslocation state. The power stroke model emphasizes the correlation between pyrophosphate release are highly analogous (Fig. 3A). As suggested by the C1 structures in the current study, the three sequential events of initial base-pairing, ribose and the required conformational changes to convert the pre- hydroxyl-induced conformational change around D238 (hereafter the translocation state to the posttranslocation state in a single and “ribose–Asp switch”), and the alignment of NTP triphosphate and largely irreversible step. Although we are not trying to reconcile D233 around the two metal ions (hereafter the “metal–Asp switch”) these two models here, our structures suggest that the RdRP likely provide the fidelity check points. The precise placement of the translocation process uses essential features of both. As suggested

Shu and Gong PNAS | Published online June 23, 2016 | E4011 Downloaded by guest on September 29, 2021 Fig. 5. A previously unidentified translocation in- termediate supports the asymmetric movement of the template–product duplex during translocation. (A) Stereo-pair images of the translocation in-

termediate complex C3S6 with a composite SA omit electron density map (contoured at 1.2 σ) of RNA, py- rophosphate (PPi), and residue D238 overlaid. The

coloring scheme is as in Fig. 3A.TheC3S4/5 pre- translocation (dark gray) and C3S1 posttranslocation (brown) complexes are shown with polymerases superimposed on the intermediate complex for com- parisons. (B) RNA-only comparison of the translocation intermediate (Left: template in cyan; Right: product in green) and the pretranslocation complex (dark gray) in the same NAC. (C) Schematic illustration of the different protein–RNA interactions in the pretran-

slocation C3S4/5, intermediate C3S6, and posttrans- location C3S1 complexes. RNA movement of the C3S6 structure was estimated using the base, ribose, and phosphate components of the pre- and post- translocation complexes as references. The zig- zagged red symbol indicates the irregular backbone conformation of the template −2 position. Phos- phate, ribose, and base are shown as circles, penta- gons, and blocks, respectively. Solid arrows indicate hydrogen bonding, electrostatic, or hydrophobic in- teractions. Gray fonts indicate weaker interactions (judged by distance) compared with interactions in- volving the same residue in other structures. Underlining indicates a change of interaction part- ner(s) for a polymerase residue when switching from the posttranslocation complex to the intermediate complex. Strikethroughs indicate nonexistent inter- actions.

by the C3S6 structure, the postcatalysis product strand is subject to +2 region in the posttranslocation state could be regarded as to Brownian motion (fast equilibrium) between the pre- the ratchet to prevent back-translocation (Fig. 6B). In the C3S6 and posttranslocation states because of the evenly distributed structure, pyrophosphate is present with a refined occupancy of interactions with the polymerase (Figs. 5 and 6B). In contrast, 0.55, comparable to the 0.66–0.72 occupancies seen in the the movement of the template strand −2to+2 region toward pretranslocation structures (Figs. 3A and 5A), but pyrophos- the upstream direction requires overcoming the sharp back- phate is totally absent both in the S1 structures in this study and in bone turn at +1/+2 and the special conformation of the −2 previously determined picornavirus polymerase EC S1 structures phosphate linkage. This movement is likely the rate-limiting (10, 14). Thus, the release of pyrophosphate may coincide with step during translocation and therefore is largely irreversible. the final upstream movement of the template −2to+2 region, Therefore, the newly established protein interactions at the −2 although whether and how the pyrophosphate release and the

E4012 | www.pnas.org/cgi/doi/10.1073/pnas.1602591113 Shu and Gong Downloaded by guest on September 29, 2021 conformational changes controlling the templates strand movement Materials and Methods PNAS PLUS are mechanically related is not obvious. Cloning and Protein Expression. The EV71 3Dpol gene within the DNA clone of It is plausible that the asymmetric movement of the two RNA SK-EV006-LPS1 (GenBank accession no. AB550335.1, genotype B) was cloned strands observed in the current study also may be a general feature of into a pET26b-Ub vector (45). The resulting plasmid was transformed into Escherichia coli strain BL21(DE3) pCG1 (kindly supplied by Craig Cameron, other classes of polymerases. Although the special backbone confor- pol − – Pennsylvania State University, State College, PA) for expression of 3D with a mation of the template 2ribosephosphate linkage is observed only C-terminal hexa-histidine tag as described previously (3, 45). 3Dpol was produced in viral RdRP–RNA complexes (2, 10, 14, 23), the sharp turn at the as a ubiquitin-fused protein, and the ubiquitin was cleaved in vivo by a coex- +1/+2 junction of the template strand is shared by several classes of pressed ubiquitin-specific carboxyl terminal protease Ubp1 to produce the 3Dpol polymerases (28, 29, 40, 41). The polymerases need to create the with homogenous native N-terminal glycine residue. Cells were grown at 25 °C active site at the downstream end of the +1 site, and therefore overnight in LB medium with 50 μg/mL kanamycin (KAN50) and 17 μg/mL the helical trace of the template strand backbone must deform around chloramphenicol (CHL17) until the OD600 was 1.0. The overnight culture was used to inoculate 1 L of LB medium with KAN50 and CHL17 to reach an initial the +1/+2 junction, making the postcatalysis translocation around the OD600 around 0.025. The cells were grown at 37 °C to an OD600 of 0.6 and then junction less fluid. Very interestingly, the eukaryotic RNA Pol II ini- were cooled to room temperature. Isopropyl-β-D-thiogalactopyranoside was tiation complex (IC) has been captured with a template–product du- added to a final concentration of 0.5 mM, and the cells were grown for an plex adopting a similar asymmetric conformation when the transcript additional 11–12 h before harvesting. – length is 4 5 nt (42). Note that these Pol II IC complexes are not pol translocation intermediates, because the asymmetric conformation Purification of EV71 3D . Cell lysis, subsequent purification, and storage procedures were as described previously (3), except that a HiTrap Q HP column (GE Healthcare) was achieved through slippage-mode movement of the product strand was used in the second chromatographic purification step. This column was equili- in the downstream direction. As a result, the −1and−2(“i-1” and brated with a buffer containing 50 mM NaCl, 25 mM Tris (pH 8.5), 0.1 mM EDTA,

“i-2” in Pol II nomenclature) nucleotides in the product strand seem 20% (vol/vol) glycerol, and 0.02% (wt/vol) NaN3, and the protein was eluted by a to form mismatches with the −1and+1 nucleotides in the template linear gradient to 600 mM NaCl. The final buffer condition for protein storage strand. These obvious conformational dynamics of the template– was 300 mM NaCl, 5 mM Tris (pH 7.5), 0.02% (wt/vol) NaN3, and 5 mM Tris (2-carboxyethyl)phosphine. The molar extinction coefficient for 3Dpol pro- product duplex may be explained in part by the intrinsic instability of a tein was calculated based on protein sequence using the ExPASy ProtParam DNA-dependent RNA polymerase IC with a short transcript. In fact, program (www.expasy.org/tools/protparam.html). The yield is typically in a similar conformation has not been observed in Pol II IC structures the range of 8–15 mg of pure protein per liter of bacterial culture. with longer transcripts or in EC structures that have established in- tensive transcript–template and transcript–polymerase interactions (29, RNA Preparation. The template strand RNA to assemble the r5 construct 42–44). Therefore, the mechanism for achieving a similar asymmetric (14) solely used in the current study was obtained by in vitro T7 RNA poly- – merase transcription using a parental plasmid pRAV23 (kindly supplied by template product conformation in these Pol II ICs appears to be Jeffrey Kieft, University of Colorado Denver, Denver, and Robert Batey, different from that suggested by the translocation intermediate in the University of Colorado Boulder, Boulder, CO) and approaches modified from current study. More high-resolution translocation-intermediate struc- protocols described previously (10, 46, 47). The 10mer RNA primer (P10) was tures are needed to refine further the understanding of the polymerase purchased from Integrated DNA Technologies. The procedures for the self-

translocation in general. As mentioned, capturing translocation in- annealing of the template strand, the subsequent annealing with P10, and BIOCHEMISTRY termediates by crystallography is somewhat serendipitous, but perhaps the r5 construct storage were as previously described (14). moleculardynamicsapproachescouldserveasavalidtoolfortesting EC Assembly, Purification, and Storage. EC assembly, purification, and storage whether other classes of polymerases also exhibit asymmetric RNA- was carried out using the protocols in the PV work (10), except that KCl was strand movements during translocation. provided at a concentration of 70 mM, the NaCl concentration was reduced

Fig. 6. Working models for viral RdRP elongation NAC and translocation. (A) The NAC model shown in a circle format. Previously reported native PV RdRP EC and three of its derivative structures obtained by CTP, 3′-deoxy-CTP (3dCTP), or ddCTP soaking serve as the reference states 1, 2, 4, and 5 (gray fonts; PDB ID codes are listed). All seven structures reported in the current study obtained by natural NTP soaking were assigned at corresponding positions in the cycle. A norovirus (NV) polymerase–RNA–CTP complex exhibiting a precatalysis closed-conformation active site represents reference state 3. (B) A schematic free

energy diagram for translocation. The pretranslocation state could establish fast equilibrium with the S6 intermediate state, and the subsequent transition to the posttranslocation state 1 of the next NAC is rate-limiting. Empty triangles indicate the interactions needed to maintain the irregular backbone con- formation of the template −2 position and the +1/+2 bend of the template. These interactions include those between the motif G T114–S115 backbone and the +1/+2 junction of the template strand backbone and those between pinky finger K127 and R188 side chain and the template strand backbone phosphates stabilizing the irregular conformation of the ribose–phosphate linkage at the −2 position. These interactions must be broken (indicated by the unlocked symbol) during the final step toward the posttranslocation state. P, product; T, template; T1/T2, transition states.

Shu and Gong PNAS | Published online June 23, 2016 | E4013 Downloaded by guest on September 29, 2021 to 40 mM for the assembly reaction, and the Hepes pH was increased to 7.0 2Fo-Fc electron density maps were generated using CNS (53). In this process, the for both the assembly reaction and complex storage. entire asymmetric unit was first divided into small boxes, each including a frac- tion not exceeding 5% of the model. For each box, the model in the box was EC Crystallization and NTP Soaking of the EC Crystals. The EC crystals were omitted for calculating the corresponding omit maps. The composite map then grown by sitting-drop vapor diffusion at 16 °C using 7.8 mg/mL EC sample. Crystals was generated by stitching all the individual omit maps together in order to grew in 1–2 wk with a precipitant solution containing 0.17 M ammonium sulfate, make the entire map, not only a specific region, less model-biased. Unless oth- 0.085 M Mes (pH 6.5), 25.5% (wt/vol) PEG 5000 monomethyl ether, and erwise indicated, all polymerase superimpositions were done using the maximum 15% (vol/vol) glycerol. NTP-soaking experiments were done under the pre- likelihood-based structure superimpositioning program THESEUS (21). cipitant solution using 5 mM NTP and 10 mM MgCl2. For each NAC complex obtained, the NTP combination and incubation time are listed in Tables 1 and 2. ACKNOWLEDGMENTS. We thank Dr. Olve Peersen for helpful discussions and valuable input on the manuscript content; Dr. Zhiyong Lou for providing Crystallographic Data Processing and Structure Determination. All final dif- the cloning material for the EV71 polymerase gene; Dr. Craig Martin and fraction data for all crystals were collected at the Shanghai Synchrotron Radiation Dr. Zhongzhou Chen for critical reading of the manuscript; Dr. Hanzhong Facility (SSRF) beamline BL17U1 at 100 Kelvin [wavelengths: 0.9791 Å for Protein Wang, Dr. Bo Zhang, and Dr. Huimin Yan for help in initiating this EV71 RdRP Data Bank (PDB) entries 5F8G and 5F8L; 0.9789 Å for PDB entries 5F8H, 5F8I, project; Wei Shi for contributions in optimizing the EC assembly reaction con- dition; Liu Deng for laboratory assistance; the Shanghai Synchrotron Radiation 5F8M, and 5F8N; and 0.9792 Å for PDB entry 5F8J]. Data (100–150°) were typi- Facility (beamline BL17U1, Shanghai, China) and the Beijing Synchrotron Ra- cally collected in 0.5° oscillation steps. Reflections were integrated, merged, and diation Facility (beamline 3W1A, Beijing, China) synchrotrons for access to scaled using HKL2000 or D*Trek v9.9 (48, 49). The initial structure solution was beamlines; and the Core Facility and Technical Support of the Wuhan Institute obtained using the molecular replacement program PHASER (50) using coordi- of Virology for access to instruments. This work was supported by National Key nates derived from the PV EC structure (PDB ID code 3OL6) as the search model Basic Research Program of China Grant 2013CB911100, National Natural Sci- (10). Manual model building and structure refinement were done using Coot and ence Foundation of China Grant 31370198, and the Hundred Talents Program Phenix, respectively (51, 52). The 3,500-K composite simulated-annealing omit of the Chinese Academy of Sciences.

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E4014 | www.pnas.org/cgi/doi/10.1073/pnas.1602591113 Shu and Gong Downloaded by guest on September 29, 2021