Crystal structure of the DENR-MCT-1 complex revealedzinc-binding site essential for heterodimer formationIvan B. Lomakina,1, Sergey E. Dmitrievb, and Thomas A. Steitza,c,2

aDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114; bBelozersky Institute of Physico-Chemical Biology,Lomonosov Moscow State University, 119234 Moscow, Russia; and cHoward Hughes Medical Institute, Yale University, New Haven, CT 06520-8114

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved November 29, 2018 (received for review June 5, 2018)

The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support nonca-nonical translation initiation, promote translation reinitiation on aspecific set of mRNAs with short upstream reading frames, andregulate ribosome recycling. DENR and MCT-1 form a heterodimer,which binds to the ribosome. We determined the crystal structure ofthe heterodimer formed by human MCT-1 and the N-terminal do-main of DENR at 2.0-Å resolution. The structure of the heterodimerreveals atomic details of the mechanism of DENR and MCT-1 inter-action. Four conserved cysteine residues of DENR (C34, C37, C44,C53) form a classical tetrahedral zinc ion-binding site, which pre-serves the structure of the DENR’s MCT-1–binding interface that isessential for the dimerization. Substitution of all four cysteines byalanine abolished a heterodimer formation. Our findings elucidatefurther the mechanism of regulation of DENR-MCT-1 activities inunconventional translation initiation, reinitiation, and recycling.

protein synthesis regulation | translation initiation | translationreinitiation | translation recycling | zinc binding

Translation initiation is the most regulated step of the proteinsynthesis. In eukaryotes, it is coordinated by more than a

dozen initiation factors (eIF) consisting of more than 30 proteinscompared with only three IFs in bacteria. Initiation factors facil-itate selection of the initiator tRNA (tRNAi

Met), the recruitmentof mRNA, and the scanning of its 5′ untranslated region (UTR) tolocate the start codon (AUG) of the main ORF, and, finally, thejoining of the small (40S) and large (60S) ribosomal subunits,which results in the formation of the 80S ribosome primed for aprotein synthesis (1, 2). Recent data have revealed that many 5′UTRs may have one or more AUG codons upstream of the mainstart codon. This may lead to a synthesis of peptides encoded bythe upstream ORFs (uORFs) or a different isoform of the mainprotein if the upstream AUG is in-frame with the main AUG.uORFs may inhibit translation of the main ORF or regulate it byreinitiation (3, 4). Reinitiation may occur if the 40S ribosomalsubunit does not dissociate from mRNA after translation termi-nation, i.e., is not recycled and is able to reach the nearest AUG.Canonical translation initiation factors (eIF1, eIF1A, eIF2, eIF3,eIF4F) are likely involved in this process; however, the exactmechanism of reinitiation is still not well understood (5, 6).The density-regulated protein (DENR) and the malignant

T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein wererecently shown to promote reinitiation after short uORFs of aspecific set of mRNAs, which are involved in cell proliferationand signaling in flies (7). The oncoprotein MCT-1 was identifiedin human T cell leukemia and lymphoma, and it has been as-sociated with increased cell proliferation and genome instability(8, 9). Synthesis of DENR protein is up-regulated with increasingcell density, and it is also overexpressed in breast and ovariancancers (10, 11). DENR forms a heterodimer with MCT-1, bothin vivo and in vitro, and this heterodimerization is essential forthe DENR-MCT-1 activity in mRNA translation initiation,reinitiation, and the ribosome recycling (12–16). Recently, wehave characterized the DENR-MCT-1 interaction with the hu-man 40S ribosomal subunit by determining the crystal structure

of the ribosomal complex with DENR-MCT-1 at 6-A resolution(13). It showed that the structure of the C-terminal domain ofDENR (C-DENR) is similar to that of the canonical translationinitiation factor 1 (eIF1), which controls the fidelity of trans-lation initiation and scanning. Moreover, C-DENR binds the 40Sribosomal subunit at the same site as eIF1. These data suggest asimilar mechanism for DENR-MCT-1 and eIF1 in discriminatingthe initiator tRNA in the P site of the 40S subunit, which iscrucial for translation initiation, reinitiation, and ribosomalrecycling (13, 17). Binding of the MCT-1 to the 40S subunit ismutually exclusive with the binding of both eIF3a and 3b sub-units of eIF3, which suggests that DENR-MCT-1 may functionas the eIF3 sensor directing the posttermination 40S subuniteither for reinitiation (if eIF3 is bound) or recycling (if eIF3 isdissociated) (13). The function of DENR-MCT-1 in translationis similar to that of the noncanonical translation initiation factoreIF2D (13, 15, 16, 18, 19). eIF2D is a single polypeptide, whichshares domain architecture with DENR-MCT-1 (Fig. 1A). Re-cent X-ray crystallography and cryo-electron microscopy (cryo-EM) studies have revealed that the C-terminal domains of botheIF2D and DENR have the same SUI1 (eIF1-like) fold andbinding site on the 40S ribosomal subunit (13, 15, 19). N-terminaldomains of eIF2D and MCT-1 also have a similar fold and interactwith the 40S subunit at the same site (13, 15, 20). Moreover,structure of the human 40S ribosomal subunit complex with eIF2D,the initiator tRNA, and the hepatitis C virus internal ribosome entry

Significance

Protein synthesis or mRNA translation by ribosomes is essen-tial for the cell’s survival. A multitude of human diseases arethe direct result of disruption of translation, specifically at theinitiation step. The density-regulated protein (DENR) and themalignant T cell-amplified sequence 1 (MCT-1/MCTS1) onco-protein support noncanonical translation initiation, reinitia-tion, and ribosome recycling linked to cancer, neurologicaldisorders, and viral infections. Here, we present the crystalstructure of a DENR-MCT-1 heterodimer, which reveals atomicdetails of DENR and MCT-1 interactions that are crucial forunderstanding their function in translation. Our results providefoundation for the future research of the mechanism of regu-lation of noncanonical protein synthesis and may potentiallybe used for antiviral, anticancer, and neurological drug design.

Author contributions: I.B.L. and T.A.S. designed research; I.B.L. performed research; I.B.L.,S.E.D., and T.A.S. analyzed data; and I.B.L., S.E.D., and T.A.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The crystallography, atomic coordinates, and structure factors have beendeposited in the Protein Data Bank, www.wwpdb.org (PDB ID 6MS4).1To whom correspondence should be addressed. Email: ivan.lomakin@yale.edu.2Deceased October 9, 2018.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1809688116/-/DCSupplemental.

Published online December 24, 2018.

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site RNA determined by cryo-EM revealed how eIF2D interactswith the P-site tRNA (15). Homology models of the MCT-1–likedomain of eIF2D and tRNA fitted in the cryo-EM map at 6.8 and9.4 A local resolution, respectively, position the CCA 3’ end of thetRNA (CCA tail) in contact with the pseudouridine synthase andarchaeosine transglycosylase (PUA) domain of eIF2D. The SWIB/MDM2 and eIF1-like domains of eIF2D interact with the ac-ceptor and D-stem of tRNA, respectively. These interactions tiltP-site tRNA toward the E site and stabilize it in the novel hybridP/E-like state (15). Does DENR-MCT-1 interact with the P-sitetRNA in a similar manner? The only data available is the cryo-EMmap of the DENR-MCT-1 translation initiation complex, whichwas of insufficient resolution (10.9 A) to model these interactions.However, M. Weisser et al. suggested interactions similar to eIF2D

based on the location within this map of the electron density at-tributed to the P-site tRNA. Interestingly, no connection was seenbetween the areas of the map attributed to the eIF1-like domain ofDENR and MCT-1 (15). Indeed, little is known about the in-teraction between DENR and MCT-1, although they are active intranslation as a heterodimer. We have recently demonstrated by thepull-down assay in vitro that the N-terminal region of DENR (N-DENR, amino acid residues 11–98) binds MCT-1. We mapped theDENR-MCT-1–binding site on the PUA domain of MCT-1, basedon the location of the unbiased electron density in the map of thehuman 40S ribosomal subunit in complex with DENR-MCT-1 (13).We proposed that this electron density, attributed to N-DENR andthe PUA domain of MCT-1, provides an interface to the binding ofinitiator tRNA. However, atomic details of interaction between

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Fig. 1. Overview of the DENR-MCT-1 heterodimer structure. (A) Domain structure of eIF2D, MCT-1, and DENR. Residues for the borders of the domains arenumbered. (B) T7•Tag antibody agarose-binding assay. T7-tagged DENR (marked as D) deletion mutants were immobilized on T7•Tag antibody agarose in thepresence of BSA and MCT-1 (marked as M) and washed with the buffer, and the bound proteins were analyzed by polyacrylamide gel electrophoresis. T7-taggedeIF1 (lane 1) was used as a negative control. DENR positions are marked by black arrows with numbers, which correspond to the amino acid residue numbers ofDENR. (C) As in B, C-DENR was used as a negative control. Coexpressed with MCT-1 and eluted from the Ni-NTA agarose N-DENR (lane 6), N-DENR (4Cys-to-Alamutant, lane 7) and N-DENR (C37Y, lane 11). Soluble (lane 8), insoluble (lane 9), and Ni-NTA agarose column flow-through proteins (lane 10) of coexpressed N-DENR(C37Y). (D) Cartoon representation of the crystal structure of the human DENR-MCT-1 heterodimer. DENR shown in coral, MCT-1 in green, and Zn2+ ion ingray. The PUA and DUF1947 domains of MCT-1 are marked. The anomalous difference Fourier map (blue mesh) around the Zn2+ ion (unique outstanding peak,σ = 20.8) is contoured at σ = 13. (E) Superposed (by DUF1947) main chains of DENR-MCT-1: DENR-MST-1 presented in this paper (colored as in B), MCT-1 alone (PDBID 3R90, red), PDB ID 5ONS (brown, blue), on ribosome (PDB ID 5VYC, gray), MCT-1-like domain of eIF2D on ribosome (PDB ID 5OA3, violet).

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DENR and MCT-1 are missing due to the low resolution (6–10 A)of the available data (13, 15).To understand how DENR interacts with MCT-1 and to elu-

cidate further the mechanism by which DENR-MCT-1 regulatestranslation initiation, reinitiation, and ribosomal recycling, wedetermined the crystal structure of the N-DENR-MCT-1 complexat 2.0-Å resolution. The structure revealed the DENR-MCT-1–binding interface of 840 Å2. We built a structural model for theamino acid residues 26–69 and identified four conserved Zn2+-boundcysteine residues of DENR that are essential for the structure ofthe MCT-1–binding site of DENR. We also determined the aminoacid residues, which form the DENR-MCT-1–binding interfaceand may contribute to the binding of the CCA tail of the tRNAbound to the mRNA in the ribosomal P site.

Results and DiscussionHeterodimer Design and Structure Determination. Previously, wedetermined that the N-terminal domain of DENR (amino acidresidues 11–98) binds to MCT-1 (13). To minimize the length ofthe N-DENR, we designed, expressed and analyzed N-terminaldeletion constructs and found that N-DENR26-98 interacts withMCT-1 (Fig. 1 B and C). Indeed, when N-DENR26-98 was coex-pressed with MCT-1, it formed a heterodimer inside the cell. Theheterodimer was purified and then crystalized by the vapor diffu-sion method. Its structure was determined by X-ray crystallographyas described in Materials and Methods (Fig. 1D and SI Appendix,Fig. S1). The crystal belongs to the P41212 tetragonal space groupand contains one DENR-MCT-1 heterodimer per asymmetric unit.A complete data set was collected to 2.0-A resolution (SI Appendix,Table S1). Initial phases were determined using the molecular

replacement method with the previously determined crystal struc-ture of MCT-1 as a search model (20). The Fo-Fc electron densitymap (Fo and Fc denote observed and calculated amplitudes, re-spectively) revealed the position of the N-DENR and also a strong,outstanding unbiased peak, which we attributed to a Zn2+ ionbased on the nearby position of the four cysteine amino acid res-idues. We then collected anomalous diffraction data at the Znabsorption edge and confirmed the bound Zn2+ ion using theanomalous difference Fourie map (Fig. 1D). The quality of themap allows us to build the region of N-DENR from the amino acidresidue 26 to 69. The electron density for the rest of the N-DENR(amino acid residues 70–98) is missing, possibly due to the flexi-bility of this region. The final model was refined at a 2.0 A reso-lution to a Rfree of 27.1% (SI Appendix, Table S1).

Overview of the N-DENR26-98-MCT-1 Heterodimer Structure. Thestructure of MCT-1 bound to DENR is slightly different from thepreviously determined structure of MCT-1 alone (Fig. 1E) (20).It is important to note that free MCT-1 was crystalized as adodecamer (in the asymmetric unit), which never was observedin the solution (20). Unusual oligomerization and also crystalpacking might affect conformation of MCT-1 in that case. In thestructure of MCT-1 presented here, both the PUA domain andthe globular N-terminal domain (DUF1947) are rotated towardeach other by about 10° and are stapled by N-DENR (Figs. 1Dand 2A). Similar conformation of an MCT-1–like domain wasseen in the structure of eIF2D bound to the 40S ribosomalsubunit and initiator tRNA determined by the cryo-EM (Fig. 1E)(15). When this paper was under review, the structure of thetruncated DENR24-51-MCT-1 heterodimer was published (21).

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Fig. 2. The DENR-MCT-1–binding interface. (A) Sur-face representation of the DENR-MCT-1 heterodimer.The surface of the area surrounding the Zn-bindingcysteines is transparent. DENR shown in coral, MCT-1in green, and Zn2+ ion in gray. The PUA and DUF1947domains of MCT-1, cysteine residues, and Zn ion aremarked. Some amino acid residues are removed for abetter view. The Zn2+-Cys bond lengths for Cys34,-37, -44, -53 are 2.31, 2.23, 2.31, and 2.36 Å, re-spectively. (B) The multiple alignment of the aminoacid sequences of the Zn-binding domain of N-DENRfrom various organisms. Only genus names are shown(for complete species names and accession numbers,see Materials and Methods). Conserved positions arehighlighted in blue. The alignment was generated byClustal Omega (www.clustal.org/omega/) algorithm.

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This structure and the one presented here are almost identical(rmsd of 0.694 for all residues; Fig. 1E and SI Appendix, Fig. S2).In the structure of the DENR-MCT-1 complex with the 40S ri-bosomal subunit, the PUA and DUF197 domains of MCT-1 arerotated toward each other even further, which may be due to anadditional restraint provided by the binding of the C-terminaldomain of DENR to the 40S subunit (Figs. 1E and 5B) (13,15, 20). The small differences in the positions of the N and Ctermini of MCT-1 were attributed either to the effect of thecrystal packing or the absence/presence of the six histidine aminoacid residues at the C terminus.The structure of the N-DENR bound to MCT-1 fits well the

unbiased electron density assigned to the N-DENR in our recentmap of the DENR-MCT-1 complex with the human ribosome(SI Appendix, Fig. S3) (13). It comprises the N terminus (aminoacid residues 26–33), followed by the globular, Zn-binding do-main (amino acid residues 34–69). The fold of this domain isstabilized by four Zn2+-bound cysteine residues, which are con-served (Figs. 1D and 2). The N terminus of DENR binds to theDUF1947 domain of MCT-1 in the vicinity of its helix α4, whilethe Zn-binding domain of DENR interacts with the PUA do-main of MCT-1 (Fig. 1D). The shape of the MCT-1–bindingsurface of DENR complements the shape of the interdomaincavity of MCT-1 (Fig. 2A). The size of the DENR-MCT-1–binding surface is 840 Å2 and is formed mostly by hydrophobicamino acid residues, which constitute two hydrophobic patcheseither on DENR or MCT-1 (Figs. 3 and 4). This agrees well withour observation that the DENR-MCT-1 heterodimer is stableeven in a 1-M concentration of salt. In addition, binding inter-actions include hydrogen bonding between amino acid residuesof the N terminus of DENR and the linker connecting DUF1947and PUA domains of MCT-1 (N of Leu29 with O of Lys87), aswell as the Zn-binding domain of DENR and the PUA domainof MCT-1 (Fig. 4 and SI Appendix, Table S2).

Zn-Binding Site. Upon building the model of the N-DENR poly-peptide chain, we observed that four cysteine residues—Cys34,-37, -44, and -53—surround a strong peak of additional electrondensity, which corresponds to a Zn2+ ion as we confirmed later(Fig. 1D andMaterials and Methods). Zn-binding proteins are the

most abundant metalloproteins in nature (22). Zn can constitutea catalytical center in enzymes and be the center of the structuralfoundation governing a protein’s fold and binding interfaces(23). Among Zn2+-binding amino acid residues such as cysteine,histidine, aspartate, and glutamate, cysteine is the most commoncoordinating ligand in proteins. The presence of four co-ordinating ligands is required for the minimal stable Zn co-ordination sphere. As it is also observed here, four cysteines in atetrahedral geometry represent one of the most common Zn2+

coordinations (23, 24). The four conserved cysteine amino acidresidues of the N-DENR—Cys34, -37, -44, and -53—are boundto Zn2+ and constitute the Zn-binding domain of N-DENR(amino acid residues 33–60), which interacts with the PUA do-main of MCT-1 mostly through hydrophobic (patch 1) andelectrostatic interactions (Figs. 2 and 3). Simultaneous mutationsof these four cysteine residues for alanine residues abolished thebinding of DENR to MCT-1 (Fig. 1C, lane 7). This demonstratesthat proper folding of the N-DENR’s Zn-binding domain iscrucial for the formation of the DENR-MCT-1 heterodimer.Interestingly, the C37Y mutation was identified in a patient withautism spectrum disorder (25). However, coimmunoprecipita-tion experiments performed in HEK293T cells showed that thismutation does not prevent DENR-MCT-1 heterodimer forma-tion (12). In agreement with this, MCT-1 was copurified with N-DENR(C37Y) when both were coexpressed in Escherichia coli.However, a large portion of the expressed N-DENR and MCT-1was insoluble, and only about 30% of soluble MCT-1 wasretained bound to N-DENR (Fig. 1C, lanes 8–11). These datasuggest that the C37Y mutation affects DENR’s stability and theconformation of the MCT-1–binding site, which likely perturbthe heterodimer equilibrium in the cell and may limit its avail-ability for interaction with the 40S ribosomal subunit. Ourstructure analysis may explain this observation. We propose thatmutation C37Y may slightly disturb the conformation of the

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Fig. 3. Surface representation of DENR-MCT-1. (A) Hydrophobic interactions.The surface formed by hydrophobic amino acid residues is colored yellow.DENR is shown in coral and MCT-1 in green. (B) Electrostatic interactions. Theelectrostatic potentials were calculated by APBS software and mapped to thesolvent-accessible surface. The intensity of color is proportional to the localpotential. HP, hydrophobic patch; RBS, ribosome-binding site.

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Fig. 4. The DENR-MCT-1 binding. N-DENR (colored in coral in cartoon rep-resentation; only amino acid residues 26–53 are shown) is bound to MCT-1(colored in green, surface representation). Atomic details are shown inmagnified panels. Zn2+ ion, O, N, and S are shown in gray, red, blue, andyellow, respectively. Some amino acid residues are removed for a betterview. Hydrogen bonds (2.6–3.8 Å) are shown by yellow dotted lines.

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37–43 region of DENR, but the folding of the Zn-binding domaincan be partially preserved because proline 40 and tyrosine 43 willprovide sufficient rigidity for the DENR-MCT-1–binding site(Fig. 2A). Both Pro40 and Tyr43 are deeply buried in the hy-drophobic pocket of the PUA domain of MCT-1 (patch 1, Fig.3A), and Tyr43 makes hydrogen bond interaction with thebackbone carbonyl oxygen of His141 of MCT-1 (Fig. 4 and SIAppendix, Table S2). In addition, the C37Y mutation may disturbthe interaction of DENR-MCT-1 with the P-site tRNA on theribosome, which would change heterodimer activity in trans-lation. We have proposed recently that this solvent-exposed re-gion of the Zn-binding domain of DENR, together with the PUAdomain of MCT-1, constitutes the surface that may interact withthe acceptor stem of the P-site tRNA (13), although the precisemechanism of the interaction between DENR-MCT-1 andtRNA remains unclear. Thus, the mutation C37Y may affect therate of the tRNAi

Met accommodation for the initiation or rein-itiation steps, as well as the rate of the deacylated P-site tRNAdissociation at the stage of ribosomal recycling. However, ourstudy does not exclude the possibility that the C37Y mutationmay change activities of DENR or DENR-MCT-1 outside of theprotein synthesis pathway. Elimination of one cysteine residuefrom the four-cysteine Zn-binding site will decrease the DENR’saffinity to the Zn2+. This may lead to complete Zn2+ removal fromDENR in the case of zinc deficiency inside the cell becauseDENR cannot compete for Zn2+ now with the majority of theother cellular Zn-binding proteins. Therefore, the mutation C37Ymay cause the collapse of the Zn-binding domain of DENR whenthe cellular concentration of Zn is too low and temporarily de-activate DENR and the DENR-MCT-1 heterodimer.

Heterodimer Interface and tRNA Binding. It was shown that DENRforms a heterodimer with MCT-1 in vivo, and, as a heterodimer,they interact with the ribosome (14, 26). Without DENR bound, thesolvent-exposed surface of MCT-1 has a positively charged regionstretched from the PUA to the DUF1947 domain, which may in-teract nonspecifically with the negatively charged tRNA or rRNAbackbone (Fig. 3B). Formation of the DENR-MCT-1 heterodimermay prevent these nonspecific interactions and ensure that theDENR-MCT-1 heterodimer is positioned specifically for the in-teraction with the tRNA bound to the P-site of the 40S ribosomalsubunit. This model proposes that the C-terminal domain of DENRinteracts with the tRNA directly in the P-site of the 40S ribosomalsubunit (13, 15). However, the mode of DENR’s N-terminal

domain and MCT-1 interaction with the acceptor stem and theCCA tail of the tRNA remains elusive. Indeed, insufficient reso-lution of the cryo-EM map of the DENR-MCT-1 translation initi-ation complex did not allow a structural model to be built.Nevertheless, M. Weisser et al. proposed that the P-site tRNA islocated near the SWIB/MDM2 and PUA domains of DENR andMCT-1, respectively, which is similar to that in the translation ini-tiation complex with eIF2D (Fig. 5A) (15). In that complex, the β2-loop of the eIF1-like domain of eIF2D interferes with the positionof the D-stem of the tRNA, the SWIB/MDM2 domain binds theacceptor stem, and the PUA domain interacts with the CCA end ofthe tRNA, keeping tRNA tilted toward the ribosomal E site inthe hybrid P/E-like state (Fig. 5A) (15).Two key structural features of eIF2D that are crucial for the

interaction with the P-site tRNA are absent in DENR: first, theβ2-loop of C-DENR is small and may not interact with the D-stemof the P-site tRNA (Fig. 5B) (13). Second, the structure of N-DENR bound to MCT-1 presented here is different from thatof the SWIB/MDM2 domain of eIF2D. In addition, electrondensity connecting C- and N- DENR (a region between aminoacid residues 70 and 110, which is less than half of the size of theSWIB/MDM2 domain of eIF2D) is not seen in the structures ofthe DENR-MCT-1 complex with the 40S subunit or N-DENR-MCT-1, which suggests that this region is flexible or unstructured.Similarly, no connection between DENR and MCT-1 werereported for the low-resolution cryo-EM map of the complex withthe tRNA, whereas the SWIB/MDM2 domain of eIF2D remainsstructured regardless of the tRNA presence (15, 19). Therefore,the absence of the interactions between DENR-MCT-1 and the P-site tRNA that force tRNA to tilt toward the E site, as proposedfor the eIF2D complex, may rather favor our recent model for theDENR-MCT-1 interaction with the P-site tRNA (13). In thismodel, the β2-loop of C-DENR does not interfere with the posi-tion of the tRNA on the ribosome, the N-DENR-MCT-1 bindingprovides the interface for the interaction with the CCA tail of theP-site tRNA, and tRNA would rather assume a conformationsimilar to the eP/I state in the canonical translation initiationcomplex (Fig. 5B) (13, 27). However, high-resolution structures ofDENR-MCT-1 translation initiation complexes are needed todistinguish between these models or to build a new one.

Conclusion. We report that the N-terminal domain of DENR26-98

binds MCT-1 through interactions between its unfolded N ter-minus and α4 helix of the DUF1947 domain of MCT-1 and

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Fig. 5. Model of eIF2D and DENR-MCT-1 interactions with the P-site tRNA. (A) Superposition of the structures of DENR-MCT-1, the eIF2D reinitiation complex(PDB ID 5OA3), and C-DENR (PDB ID 5VYC). (B) As in A, with MCT-1–like and SWIB/MDM2 domains of eIF2D removed and proposed P-site tRNA (in gold)stabilized by DENR-MCT-1 added. The 40S ribosomal subunit is shown as a gray surface, tRNA in the eIF2D reinitiation complex is in blue, eIF2D is in violet,MCT-1 is in green, and DENR is in coral. Spheres show Zn2+ ion (gray) and MCT-1 phosphorylation site (Ser118, red). Dashed line connects N- and C-terminaldomains of DENR.

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between its globular domain and the PUA domain of MCT-1.Our structure revealed that the globular domain of N-DENRincludes four cysteine amino acid residues (C34, C37, C44,C53), which are bound to the Zn2+ ion. The Zn2+ is tetrahedrallycoordinated, and it is crucial for stabilizing the tertiary structure ofthe DENR’s MCT-1–binding domain because substitution of allfor cysteines by alanines abolished DENR MCT-1 binding. Basedon our structure, we proposed an explanation for the single C37Ymutation in the Zn-binding domain of DENR, which was recentlylinked to the autism spectrum disorder (25). Mutation C37Y maypartially destabilize the DENR-MCT-1 heterodimer and/or affectthe conformation of the P-site tRNA. These will influence thedynamics of translation initiation, reinitiation, or ribosomal recy-cling. Our data provide insights into the mechanism of the non-canonical translation initiation, reinitiation, and ribosomal recyclingby demonstrating that Zn-ion binding regulates the interactionbetween DENR and MCT-1 and between the DENR-MCT-1heterodimer and the P-site tRNA in the context of the ribosomalcomplex. Additional structural and functional studies will beneeded to determine whether other proteins are involved in thisregulation and to elucidate the mechanism further.

Materials and MethodsAll DENR’s deletion mutants were made by PCR using the primers encodedthe BamHI restriction site, the tobacco etch virus protease cleavage site atthe 5′-region in frame with the DENR-encoding sequence, and the HindIIIsite at the 3′-region. PCR fragments were cloned in pET28a expression vector(Novagen). For coexpression with DENR, the MCT-1–coding region was am-plified by PCR using a 5′-region primer with an encoded XhoI site, a T7promoter and the ribosome-binding site from pET33-MCTS1 (13), and a 3′-region primer with the XhoI site. PCR fragment was then cloned in thepET28a-TEV-N-98-98) plasmid using the XhoI site. Cysteine-to-alanine mu-tations were introduced using a QuikChange Lightning Site-Directed Mu-tagenesis Kit (Agilent Technologies). Protein expression, purification, and inan vitro-binding assay were performed as described previously (13).

Crystals were grown in 24-well sitting-drop plates using the vapor diffu-sion technique. Three microliters of DENR26-98-MCT-1 (20 mg/mL) weremixed with 3 μL of reservoir solution (50 mM Tris·HCl, 20% PEG 4000, pH8.6). Plates were incubated at 20 °C for 14–19 d. Crystals were stabilized bysoaking for 15 min in the following buffer: 0.1 M of NaCl, 0.05 M of Tris·HCl,

pH 8.6, 30% of PEG 4000, and 30% of glycerol. After stabilization, crystalswere frozen in liquid nitrogen.

X-ray diffraction data collection was performed at the Advanced PhotonSource in the Argonne National Laboratory at beamline 24ID-C. A completedataset was collected from a single crystal to a 2.0-Å resolution. A single-wavelength anomalous diffraction dataset for DENR-MCT-1 was collected atthe Zn absorption peak wavelength of 1.2822 Å. Diffraction data were pro-cessed and scaled using X-ray Detector Software (SI Appendix, Table S1) (28).

The structure was solved by molecular replacement using PHASER from theCCP4 program suite and the structure of humanMCT-1 as amodel (PDB ID3R90,chain A) (20, 29). Program Coot was used for model building and Refmac fromthe CCP4i suite was used for the model refinement (29, 30). The final cross-validated Rfree after model refinement was 25.8% (SI Appendix, Table S1).

Analysis of the DENR-MCT-1–binding interface was done using PDBePISAservice at www.ebi.ac.uk/pdbe/prot_int/pistart.html (31). The electrostatic po-tentials were calculated by APBS software and mapped to the solvent-accessiblesurface (32). The intensity of color is proportional to the local potential.

Alignment of the amino acid sequences of the Zn-binding domain ofN-DENR was generated by Clustal Omega (www.clustal.org/omega/) algorithmwith default settings and visualized by the Jalview program (www.jalview.org).The sequences from the following organisms were used: Homo sapiens(NP_003668), Mus musculus (NP_080879), Gallus gallus (NP_001072973), Anoliscarolinensis (XP_003222792), Xenopus tropicalis (NP_001006814), Danio rerio(NP_001002697), Drosophila melanogaster (NP_573176), Caenorhabditis ele-gans (NP_499450), Schistosoma japonicum (AAW27105), Amphimedonqueenslandica (XP_003385869), Hydra vulgaris (XP_002165948), Dictyosteliumdiscoideum (XP_641593), Entamoeba invadens (XP_004259902), Trypanosomacruzi (EKF32067), Plasmodium vivax (XP_001617181), Schizosaccharomycespombe (NP_596803), Saccharomyces cerevisiae (NP_012548),Neurospora crassa(XP_965370), Physcomitrella patens (XP_024386006), and Arabidopsis thaliana(NP_196751). Accession numbers are shown in parentheses.

Figures showing atomic models were generated using PYMOL [DelanoScientific, The PyMOL Molecular Graphics System, Version 1.8 Schrodinger(https://www.pymol.org)].

ACKNOWLEDGMENTS. We thank Dr. Jimin Wang for his helpful critiques ofthis manuscript and discussions regarding this project. Staffs at the ArgonneNational Laboratory (Northeastern Collaborative Access Team 24ID-C) havebeen extremely helpful in facilitating X-ray data collection. This work wassupported by Russian Science Foundation Grant 18-14-00291 (to S.E.D.); bythe Howard Hughes Medical Institute; and by NIH Grant GM022778(to T.A.S.).

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