Crystal Structure of the DENR-MCT-1 Complex Revealed Zinc-Binding Site Essential for Heterodimer Formation

Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation

Ivan 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- of the ribosomal complex with DENR-MCT-1 at 6-Åresolution amplified sequence 1 (MCT-1/MCTS1) oncoprotein support nonca- (13). It showed that the structure of the C-terminal domain of nonical translation initiation, promote translation reinitiation on a DENR (C-DENR) is similar to that of the canonical translation specific set of mRNAs with short upstream reading frames, and initiation factor 1 (eIF1), which controls the fidelity of trans- regulate ribosome recycling. DENR and MCT-1 form a heterodimer, lation initiation and scanning. Moreover, C-DENR binds the 40S which binds to the ribosome. We determined the crystal structure of ribosomal subunit at the same site as eIF1. These data suggest a the heterodimer formed by human MCT-1 and the N-terminal do- similar mechanism for DENR-MCT-1 and eIF1 in discriminating main of DENR at 2.0-Å resolution. The structure of the heterodimer the initiator tRNA in the P site of the 40S subunit, which is reveals atomic details of the mechanism of DENR and MCT-1 inter- crucial for translation initiation, reinitiation, and ribosomal action. Four conserved cysteine residues of DENR (C34, C37, C44, recycling (13, 17). Binding of the MCT-1 to the 40S subunit is C53) form a classical tetrahedral zinc ion-binding site, which pre- mutually exclusive with the binding of both eIF3a and 3b sub- serves the structure of the DENR’sMCT-1–binding interface that is units of eIF3, which suggests that DENR-MCT-1 may function essential for the dimerization. Substitution of all four cysteines by as the eIF3 sensor directing the posttermination 40S subunit alanine abolished a heterodimer formation. Our findings elucidate either for reinitiation (if eIF3 is bound) or recycling (if eIF3 is further the mechanism of regulation of DENR-MCT-1 activities in dissociated) (13). The function of DENR-MCT-1 in translation unconventional translation initiation, reinitiation, and recycling. is similar to that of the noncanonical translation initiation factor eIF2D (13, 15, 16, 18, 19). eIF2D is a single polypeptide, which BIOCHEMISTRY protein synthesis regulation | translation initiation | translation shares domain architecture with DENR-MCT-1 (Fig. 1A). Re- reinitiation | translation recycling | zinc binding cent X-ray crystallography and cryo-electron microscopy (cryo- EM) studies have revealed that the C-terminal domains of both ranslation initiation is the most regulated step of the protein eIF2D and DENR have the same SUI1 (eIF1-like) fold and Tsynthesis. In eukaryotes, it is coordinated by more than a binding site on the 40S ribosomal subunit (13, 15, 19). N-terminal dozen initiation factors (eIF) consisting of more than 30 proteins domains of eIF2D and MCT-1 also have a similar fold and interact compared with only three IFs in bacteria. Initiation factors facil- with the 40S subunit at the same site (13, 15, 20). Moreover, Met itate selection of the initiator tRNA (tRNAi ), the recruitment structure of the human 40S ribosomal subunit complex with eIF2D, of mRNA, and the scanning of its 5′ untranslated region (UTR) to the initiator tRNA, and the hepatitis C virus internal ribosome entry locate the start codon (AUG) of the main ORF, and, finally, the joining of the small (40S) and large (60S) ribosomal subunits, Significance which results in the formation of the 80S ribosome primed for a ′ protein synthesis (1, 2). Recent data have revealed that many 5 Protein synthesis or mRNA translation by ribosomes is essen- UTRs may have one or more AUG codons upstream of the main tial for the cell’s survival. A multitude of human diseases are start codon. This may lead to a synthesis of peptides encoded by the direct result of disruption of translation, specifically at the the upstream ORFs (uORFs) or a different isoform of the main initiation step. The density-regulated protein (DENR) and the protein if the upstream AUG is in-frame with the main AUG. malignant T cell-amplified sequence 1 (MCT-1/MCTS1) onco- uORFs may inhibit translation of the main ORF or regulate it by protein support noncanonical translation initiation, reinitia- reinitiation (3, 4). Reinitiation may occur if the 40S ribosomal tion, and ribosome recycling linked to cancer, neurological subunit does not dissociate from mRNA after translation termi- disorders, and viral infections. Here, we present the crystal nation, i.e., is not recycled and is able to reach the nearest AUG. structure of a DENR-MCT-1 heterodimer, which reveals atomic Canonical translation initiation factors (eIF1, eIF1A, eIF2, eIF3, details of DENR and MCT-1 interactions that are crucial for eIF4F) are likely involved in this process; however, the exact understanding their function in translation. Our results provide mechanism of reinitiation is still not well understood (5, 6). foundation for the future research of the mechanism of regu- The density-regulated protein (DENR) and the malignant lation of noncanonical protein synthesis and may potentially T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein were be used for antiviral, anticancer, and neurological drug design. recently shown to promote reinitiation after short uORFs of a specific set of mRNAs, which are involved in cell proliferation Author contributions: I.B.L. and T.A.S. designed research; I.B.L. performed research; I.B.L., and signaling in flies (7). The oncoprotein MCT-1 was identified S.E.D., and T.A.S. analyzed data; and I.B.L., S.E.D., and T.A.S. wrote the paper. in human T cell leukemia and lymphoma, and it has been as- The authors declare no conflict of interest. sociated with increased cell proliferation and genome instability This article is a PNAS Direct Submission. (8, 9). Synthesis of DENR protein is up-regulated with increasing Published under the PNAS license. cell density, and it is also overexpressed in breast and ovarian Data deposition: The crystallography, atomic coordinates, and structure factors have been cancers (10, 11). DENR forms a heterodimer with MCT-1, both deposited in the Protein Data Bank, www.wwpdb.org (PDB ID 6MS4). in vivo and in vitro, and this heterodimerization is essential for 1To whom correspondence should be addressed. Email: [email protected]. the DENR-MCT-1 activity in mRNA translation initiation, 2Deceased October 9, 2018. reinitiation, and the ribosome recycling (12–16). Recently, we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. have characterized the DENR-MCT-1 interaction with the hu- 1073/pnas.1809688116/-/DCSupplemental. man 40S ribosomal subunit by determining the crystal structure

www.pnas.org/cgi/doi/10.1073/pnas.1809688116 PNAS Latest Articles | 1of6 Downloaded by guest on September 26, 2021 A 1 93 173 383 470 491 584 D DUF1947 PUA WH SWIB/MDM2 SUI1 eIF2D MCT-1 DUF1947 PUA SUI1 DENR 1 92 181 1 93 106 198 MCT-1 N-DENR PUA

B Zn 1 69 MW D11-98+M eIF1+M D110-198+M D26-198+M T7-tag M, no kDa D10-198+M D51-198+M 62- α4 49- α3

38- 10-198 26-198 51-198 DUF1947 28- MCT-1 110-198 17- 11-98 N-DENR 26 (26-98) 14- 1 2 3 4 5 6 7 C E 69 PUA D26-98+M D26-98+M D11-98+M M, reference D26-98 C37Y+M, soluble C37Y+M, insoluble cDENR+M D26-98C2A+M C37Y+M, flowthrough C37Y+M, eluate 62- N-DENR 49- 26 38- DUF1947 28- MCT-1 17- 11-98 26-98 14- C 1 2 3 4 5 6 7 8 9 10 11

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 are numbered. (B)T7•Tag antibody agarose-binding assay. T7-tagged DENR (marked as D) deletion mutants were immobilized on T7•Tag antibody agarose in the presence of BSA and MCT-1 (marked as M) and washed with the buffer, and the bound proteins were analyzed by polyacrylamide gel electrophoresis. T7-tagged eIF1 (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 of DENR. (C)AsinB, 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-Ala mutant, 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 in + gray. 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 (PDB ID 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).

site RNA determined by cryo-EM revealed how eIF2D interacts based on the location within this map of the electron density at- with the P-site tRNA (15). Homology models of the MCT-1–like tributed to the P-site tRNA. Interestingly, no connection was seen domain of eIF2D and tRNA fitted in the cryo-EM map at 6.8 and between the areas of the map attributed to the eIF1-like domain of 9.4 Ålocal resolution, respectively, position the CCA 3’ end of the DENR and MCT-1 (15). Indeed, little is known about the in- tRNA (CCA tail) in contact with the pseudouridine synthase and teraction between DENR and MCT-1, although they are active in archaeosine transglycosylase (PUA) domain of eIF2D. The SWIB/ translation as a heterodimer. We have recently demonstrated by the MDM2 and eIF1-like domains of eIF2D interact with the ac- pull-down assay in vitro that the N-terminal region of DENR (N- ceptor and D-stem of tRNA, respectively. These interactions tilt DENR,aminoacidresidues11–98) binds MCT-1. We mapped the P-site tRNA toward the E site and stabilize it in the novel hybrid DENR-MCT-1–binding site on the PUA domain of MCT-1, based P/E-like state (15). Does DENR-MCT-1 interact with the P-site on the location of the unbiased electron density in the map of the tRNA in a similar manner? The only data available is the cryo-EM human 40S ribosomal subunit in complex with DENR-MCT-1 (13). map of the DENR-MCT-1 translation initiation complex, which We proposed that this electron density, attributed to N-DENR and was of insufficient resolution (10.9 Å) to model these interactions. the PUA domain of MCT-1, provides an interface to the binding of However, M. Weisser et al. suggested interactions similar to eIF2D initiator tRNA. However, atomic details of interaction between

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1809688116 Lomakin et al. Downloaded by guest on September 26, 2021 DENR and MCT-1 are missing due to the low resolution (6–10 Å) replacement method with the previously determined crystal struc- of the available data (13, 15). ture of MCT-1 as a search model (20). The Fo-Fc electron density To understand how DENR interacts with MCT-1 and to elu- map (Fo and Fc denote observed and calculated amplitudes, re- cidate further the mechanism by which DENR-MCT-1 regulates spectively) revealed the position of the N-DENR and also a strong, + translation initiation, reinitiation, and ribosomal recycling, we outstanding unbiased peak, which we attributed to a Zn2 ion determined the crystal structure of the N-DENR-MCT-1 complex based on the nearby position of the four cysteine amino acid res- at 2.0-Å resolution. The structure revealed the DENR-MCT-1– idues. We then collected anomalous diffraction data at the Zn + binding interface of 840 Å2.Webuiltastructuralmodelforthe absorption edge and confirmed the bound Zn2 ion using the + aminoacidresidues26–69 and identified four conserved Zn2 -bound anomalous difference Fourie map (Fig. 1D). The quality of the cysteine residues of DENR that are essential for the structure of map allows us to build the region of N-DENR from the amino acid the MCT-1–binding site of DENR. We also determined the amino residue 26 to 69. The electron density for the rest of the N-DENR acid residues, which form the DENR-MCT-1–binding interface (amino acid residues 70–98) is missing, possibly due to the flexi- and may contribute to the binding of the CCA tail of the tRNA bility of this region. The final model was refined at a 2.0 Åreso- bound to the mRNA in the ribosomal P site. lution to a Rfree of 27.1% (SI Appendix,TableS1).

Results and Discussion Overview of the N-DENR26-98-MCT-1 Heterodimer Structure. The Heterodimer Design and Structure Determination. Previously, we structure of MCT-1 bound to DENR is slightly different from the determined that the N-terminal domain of DENR (amino acid previously determined structure of MCT-1 alone (Fig. 1E) (20). residues 11–98) binds to MCT-1 (13). To minimize the length of It is important to note that free MCT-1 was crystalized as a the N-DENR, we designed, expressed and analyzed N-terminal dodecamer (in the asymmetric unit), which never was observed deletion constructs and found that N-DENR26-98 interacts with in the solution (20). Unusual oligomerization and also crystal MCT-1 (Fig. 1 B and C). Indeed, when N-DENR26-98 was coex- packing might affect conformation of MCT-1 in that case. In the pressed with MCT-1, it formed a heterodimer inside the cell. The structure of MCT-1 presented here, both the PUA domain and heterodimer was purified and then crystalized by the vapor diffu- the globular N-terminal domain (DUF1947) are rotated toward sion method. Its structure was determined by X-ray crystallography each other by about 10° and are stapled by N-DENR (Figs. 1D as described in Materials and Methods (Fig. 1D and SI Appendix, and 2A). Similar conformation of an MCT-1–like domain was Fig. S1). The crystal belongs to the P41212 tetragonal space group seen in the structure of eIF2D bound to the 40S ribosomal BIOCHEMISTRY and contains one DENR-MCT-1 heterodimer per asymmetric unit. subunit and initiator tRNA determined by the cryo-EM (Fig. 1E) A complete data set was collected to 2.0-Åresolution (SI Appendix, (15). When this paper was under review, the structure of the Table S1). Initial phases were determined using the molecular truncated DENR24-51-MCT-1 heterodimer was published (21).

A MCT-1 N-DENR (26-98)

PUA 69

C53 C37

C44 Zn

Y43

26 P40

C34 DUF1947

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-binding cysteines is transparent. DENR shown in coral, MCT-1 + in green, and Zn2 ion in gray. The PUA and DUF1947 domains of MCT-1, cysteine residues, and Zn ion are marked. Some amino acid residues are removed for a + better view. The Zn2 -Cys bond lengths for Cys34, -37, -44, -53 are 2.31, 2.23, 2.31, and 2.36 Å, respectively. (B) The multiple alignment of the amino acid sequences of the Zn-binding domain of N-DENR from various organisms. Only genus names are shown (for complete species names and accession numbers, see Materials and Methods). Conserved positions are highlighted in blue. The alignment was generated by Clustal Omega (www.clustal.org/omega/) algorithm.

Lomakin et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 26, 2021 MCT-1 MCT-1 N-DENR N-DENR most abundant metalloproteins in nature (22). Zn can constitute A HP1 HP1 PUA a catalytical center in enzymes and be the center of the structural foundation 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 common coordinating ligand in proteins. The presence of four co- 180o ordinating ligands is required for the minimal stable Zn coordination sphere. As it is also observed here, four cysteines in a HP2 HP2 + tetrahedral geometry represent one of the most common Zn2 DUF1947 coordinations (23, 24). The four conserved cysteine amino acid residues of the N-DENR—Cys34, -37, -44, and -53—are bound B + to Zn2 and constitute the Zn-binding domain of N-DENR (amino acid residues 33–60), which interacts with the PUA domain of MCT-1 mostly through hydrophobic (patch 1) and electrostatic interactions (Figs. 2 and 3). Simultaneous mutations

o of these four cysteine residues for alanine residues abolished the 180 binding of DENR to MCT-1 (Fig. 1C, lane 7). This demonstrates that proper folding of the N-DENR’s Zn-binding domain is crucial for the formation of the DENR-MCT-1 heterodimer. RBS Interestingly, the C37Y mutation was identified in a patient with Fig. 3. Surface representation of DENR-MCT-1. (A) Hydrophobic interactions. autism spectrum disorder (25). However, coimmunoprecipita- The surface formed by hydrophobic amino acid residues is colored yellow. tion experiments performed in HEK293T cells showed that this DENR is shown in coral and MCT-1 in green. (B) Electrostatic interactions. The mutation does not prevent DENR-MCT-1 heterodimer forma- electrostatic potentials were calculated by APBS software and mapped to the tion (12). In agreement with this, MCT-1 was copurified with N- solvent-accessible surface. The intensity of color is proportional to the local DENR(C37Y) when both were coexpressed in Escherichia coli. potential. HP, hydrophobic patch; RBS, ribosome-binding site. However, a large portion of the expressed N-DENR and MCT-1 was insoluble, and only about 30% of soluble MCT-1 was retained bound to N-DENR (Fig. 1C, lanes 8–11). These data This structure and the one presented here are almost identical suggest that the C37Y mutation affects DENR’s stability and the E SI Appendix (rmsd of 0.694 for all residues; Fig. 1 and , Fig. S2). conformation of the MCT-1–binding site, which likely perturb In the structure of the DENR-MCT-1 complex with the 40S ri- the heterodimer equilibrium in the cell and may limit its avail- bosomal subunit, the PUA and DUF197 domains of MCT-1 are ability for interaction with the 40S ribosomal subunit. Our rotated toward each other even further, which may be due to an structure analysis may explain this observation. We propose that additional restraint provided by the binding of the C-terminal mutation C37Y may slightly disturb the conformation of the domain of DENR to the 40S subunit (Figs. 1E and 5B) (13, 15, 20). The small differences in the positions of the N and C termini of MCT-1 were attributed either to the effect of the crystal packing or the absence/presence of the six histidine amino PUA acid residues at the C terminus. C53 C44 Y46 The structure of the N-DENR bound to MCT-1 fits well the C37 Zn K99 unbiased electron density assigned to the N-DENR in our recent C34 Y46 Y43 map of the DENR-MCT-1 complex with the human ribosome Y43 SI Appendix K139 ( , Fig. S3) (13). It comprises the N terminus (amino Y33 E45 acid residues 26–33), followed by the globular, Zn-binding domain (amino acid residues 34–69). The fold of this domain is H141 + P40 stabilized by four Zn2 -bound cysteine residues, which are con- E42 served (Figs. 1D and 2). The N terminus of DENR binds to the Q140 DUF1947 domain of MCT-1 in the vicinity of its helix α4, while Y27 the Zn-binding domain of DENR interacts with the PUA do- DUF1947 main of MCT-1 (Fig. 1D). The shape of the MCT-1–binding surface of DENR complements the shape of the interdomain H176 N171 cavity of MCT-1 (Fig. 2A). The size of the DENR-MCT-1– K87 binding surface is 840 Å2 and is formed mostly by hydrophobic W175 M47 amino acid residues, which constitute two hydrophobic patches S38 either on DENR or MCT-1 (Figs. 3 and 4). This agrees well with L29

our observation that the DENR-MCT-1 heterodimer is stable C37 even in a 1-M concentration of salt. In addition, binding inter- H86 actions include hydrogen bonding between amino acid residues of the N terminus of DENR and the linker connecting DUF1947 Y33 and PUA domains of MCT-1 (N of Leu29 with O of Lys87), as H141 Y27 well as the Zn-binding domain of DENR and the PUA domain Q140 E42 of MCT-1 (Fig. 4 and SI Appendix, Table S2). F90 Fig. 4. The DENR-MCT-1 binding. N-DENR (colored in coral in cartoon rep- Zn-Binding Site. Upon building the model of the N-DENR poly- – — resentation; only amino acid residues 26 53 are shown) is bound to MCT-1 peptide chain, we observed that four cysteine residues Cys34, (colored in green, surface representation). Atomic details are shown in — + -37, -44, and -53 surround a strong peak of additional electron magnified panels. Zn2 ion, O, N, and S are shown in gray, red, blue, and 2+ density, which corresponds to a Zn ion as we confirmed later yellow, respectively. Some amino acid residues are removed for a better (Fig. 1D and Materials and Methods). Zn-binding proteins are the view. Hydrogen bonds (2.6–3.8 Å) are shown by yellow dotted lines.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1809688116 Lomakin et al. Downloaded by guest on September 26, 2021 37–43 region of DENR, but the folding of the Zn-binding domain domain and MCT-1 interaction with the acceptor stem and the can be partially preserved because proline 40 and tyrosine 43 will CCA tail of the tRNA remains elusive. Indeed, insufficient reso- provide sufficient rigidity for the DENR-MCT-1–binding site lution of the cryo-EM map of the DENR-MCT-1 translation initi- (Fig. 2A). Both Pro40 and Tyr43 are deeply buried in the hy- ation complex did not allow a structural model to be built. drophobic pocket of the PUA domain of MCT-1 (patch 1, Fig. Nevertheless, M. Weisser et al. proposed that the P-site tRNA is 3A), and Tyr43 makes hydrogen bond interaction with the located near the SWIB/MDM2 and PUA domains of DENR and backbone carbonyl oxygen of His141 of MCT-1 (Fig. 4 and SI MCT-1, respectively, which is similar to that in the translation ini- Appendix, Table S2). In addition, the C37Y mutation may disturb tiation complex with eIF2D (Fig. 5A) (15). In that complex, the β2- the interaction of DENR-MCT-1 with the P-site tRNA on the loop of the eIF1-like domain of eIF2D interferes with the position ribosome, which would change heterodimer activity in trans- of the D-stem of the tRNA, the SWIB/MDM2 domain binds the lation. We have proposed recently that this solvent-exposed re- acceptor stem, and the PUA domain interacts with the CCA end of gion of the Zn-binding domain of DENR, together with the PUA the tRNA, keeping tRNA tilted toward the ribosomal E site in domain of MCT-1, constitutes the surface that may interact with the hybrid P/E-like state (Fig. 5A)(15). the acceptor stem of the P-site tRNA (13), although the precise Two key structural features of eIF2D that are crucial for the mechanism of the interaction between DENR-MCT-1 and interaction with the P-site tRNA are absent in DENR: first, the tRNA remains unclear. Thus, the mutation C37Y may affect the β2-loop of C-DENR is small and may not interact with the D-stem Met B rate of the tRNAi accommodation for the initiation or rein- of the P-site tRNA (Fig. 5 ) (13). Second, the structure of N- itiation steps, as well as the rate of the deacylated P-site tRNA DENR bound to MCT-1 presented here is different from that dissociation at the stage of ribosomal recycling. However, our of the SWIB/MDM2 domain of eIF2D. In addition, electron study does not exclude the possibility that the C37Y mutation density connecting C- and N- DENR (a region between amino may change activities of DENR or DENR-MCT-1 outside of the acid residues 70 and 110, which is less than half of the size of the protein synthesis pathway. Elimination of one cysteine residue SWIB/MDM2 domain of eIF2D) is not seen in the structures of from the four-cysteine Zn-binding site will decrease the DENR’s the DENR-MCT-1 complex with the 40S subunit or N-DENR- + + affinity to the Zn2 . This may lead to complete Zn2 removal from MCT-1, which suggests that this region is flexible or unstructured. DENR in the case of zinc deficiency inside the cell because Similarly, no connection between DENR and MCT-1 were + DENR cannot compete for Zn2 now with the majority of the reported for the low-resolution cryo-EM map of the complex with other cellular Zn-binding proteins. Therefore, the mutation C37Y the tRNA, whereas the SWIB/MDM2 domain of eIF2D remains BIOCHEMISTRY may cause the collapse of the Zn-binding domain of DENR when structured regardless of the tRNA presence (15, 19). Therefore, the cellular concentration of Zn is too low and temporarily de- the absence of the interactions between DENR-MCT-1 and the P- activate DENR and the DENR-MCT-1 heterodimer. site tRNA that force tRNA to tilt toward the E site, as proposed for the eIF2D complex, may rather favor our recent model for the Heterodimer Interface and tRNA Binding. It was shown that DENR DENR-MCT-1 interaction with the P-site tRNA (13). In this forms a heterodimer with MCT-1 in vivo, and, as a heterodimer, model, the β2-loop of C-DENR does not interfere with the posi- they interact with the ribosome (14, 26). Without DENR bound, the tion of the tRNA on the ribosome, the N-DENR-MCT-1 binding solvent-exposed surface of MCT-1 has a positively charged region provides the interface for the interaction with the CCA tail of the stretched from the PUA to the DUF1947 domain, which may in- P-site tRNA, and tRNA would rather assume a conformation teract nonspecifically with the negatively charged tRNA or rRNA similar to the eP/I state in the canonical translation initiation backbone (Fig. 3B). Formation of the DENR-MCT-1 heterodimer complex (Fig. 5B) (13, 27). However, high-resolution structures of may prevent these nonspecific interactions and ensure that the DENR-MCT-1 translation initiation complexes are needed to DENR-MCT-1 heterodimer is positioned specifically for the in- distinguish between these models or to build a new one. teraction with the tRNA bound to the P-site of the 40S ribosomal subunit. This model proposes that the C-terminal domain of DENR Conclusion. We report that the N-terminal domain of DENR26-98 interacts with the tRNA directly in the P-site of the 40S ribosomal binds MCT-1 through interactions between its unfolded N ter- subunit (13, 15). However, the mode of DENR’sN-terminal minus and α4 helix of the DUF1947 domain of MCT-1 and

ABeIF2D MCT-1 PUA A C C tRNA P/E-like state 69

106 N-DENR SWIB/ D-stem MDM2

P site tRNA C-DENR eIF1-like eP/I state (SUI1) 198 40S 40S

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)AsinA, 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-terminal domains of DENR.

Lomakin et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 26, 2021 between its globular domain and the PUA domain of MCT-1. pH 8.6, 30% of PEG 4000, and 30% of glycerol. After stabilization, crystals Our structure revealed that the globular domain of N-DENR were frozen in liquid nitrogen. includes four cysteine amino acid residues (C34, C37, C44, X-ray diffraction data collection was performed at the Advanced Photon + + C53), which are bound to the Zn2 ion. The Zn2 is tetrahedrally Source in the Argonne National Laboratory at beamline 24ID-C. A complete coordinated, and it is crucial for stabilizing the tertiary structure of dataset was collected from a single crystal to a 2.0-Å resolution. A single- the DENR’sMCT-1–binding domain because substitution of all wavelength anomalous diffraction dataset for DENR-MCT-1 was collected at the Zn absorption peak wavelength of 1.2822 Å. Diffraction data were pro- for cysteines by alanines abolished DENR MCT-1 binding. Based cessed and scaled using X-ray Detector Software (SI Appendix,TableS1)(28). on our structure, we proposed an explanation for the single C37Y The structure was solved by molecular replacement using PHASER from the mutation in the Zn-binding domain of DENR, which was recently CCP4 program suite and the structure of human MCT-1 as a model (PDB ID 3R90, linked to the autism spectrum disorder (25). Mutation C37Y may chain A) (20, 29). Program Coot was used for model building and Refmac from partially destabilize the DENR-MCT-1 heterodimer and/or affect the CCP4i suite was used for the model refinement (29, 30). The final cross-

the conformation of the P-site tRNA. These will influence the validated Rfree after model refinement was 25.8% (SI Appendix,TableS1). dynamics of translation initiation, reinitiation, or ribosomal recy- Analysis of the DENR-MCT-1–binding interface was done using PDBePISA cling. Our data provide insights into the mechanism of the non- service at www.ebi.ac.uk/pdbe/prot_int/pistart.html (31). The electrostatic po- canonical translation initiation, reinitiation, and ribosomal recycling tentials were calculated by APBS software and mapped to the solvent-accessible by demonstrating that Zn-ion binding regulates the interaction surface (32). The intensity of color is proportional to the local potential. between DENR and MCT-1 and between the DENR-MCT-1 Alignment of the amino acid sequences of the Zn-binding domain of heterodimer and the P-site tRNA in the context of the ribosomal N-DENR was generated by Clustal Omega (www.clustal.org/omega/)algorithm complex. Additional structural and functional studies will be with default settings and visualized by the Jalview program (www.jalview.org). needed to determine whether other proteins are involved in this The sequences from the following organisms were used: Homo sapiens (NP_003668), Mus musculus (NP_080879), Gallus gallus (NP_001072973), Anolis regulation and to elucidate the mechanism further. carolinensis (XP_003222792), Xenopus tropicalis (NP_001006814), Danio rerio (NP_001002697), Drosophila melanogaster (NP_573176), Caenorhabditis ele- Materials and Methods gans (NP_499450), Schistosoma japonicum (AAW27105), Amphimedon All DENR’s deletion mutants were made by PCR using the primers encoded queenslandica (XP_003385869), Hydra vulgaris (XP_002165948), Dictyostelium the BamHI restriction site, the tobacco etch virus protease cleavage site at discoideum (XP_641593), Entamoeba invadens (XP_004259902), Trypanosoma ′ the 5 -region in frame with the DENR-encoding sequence, and the HindIII cruzi (EKF32067), Plasmodium vivax (XP_001617181), Schizosaccharomyces ′ site at the 3 -region. PCR fragments were cloned in pET28a expression vector pombe (NP_596803), Saccharomyces cerevisiae (NP_012548), Neurospora crassa – (Novagen). For coexpression with DENR, the MCT-1 coding region was am- (XP_965370), Physcomitrella patens (XP_024386006), and Arabidopsis thaliana ′ plified by PCR using a 5 -region primer with an encoded XhoI site, a T7 (NP_196751). Accession numbers are shown in parentheses. promoter and the ribosome-binding site from pET33-MCTS1 (13), and a 3′- Figures showing atomic models were generated using PYMOL [Delano region primer with the XhoI site. PCR fragment was then cloned in the Scientific, The PyMOL Molecular Graphics System, Version 1.8 Schrödinger pET28a-TEV-N-98-98) plasmid using the XhoI site. Cysteine-to-alanine mu- (https://www.pymol.org)]. tations were introduced using a QuikChange Lightning Site-Directed Mu- tagenesis Kit (Agilent Technologies). Protein expression, purification, and in ACKNOWLEDGMENTS. We thank Dr. Jimin Wang for his helpful critiques of an vitro-binding assay were performed as described previously (13). this manuscript and discussions regarding this project. Staffs at the Argonne Crystals were grown in 24-well sitting-drop plates using the vapor diffu- 26-98 National Laboratory (Northeastern Collaborative Access Team 24ID-C) have sion technique. Three microliters of DENR -MCT-1 (20 mg/mL) were been extremely helpful in facilitating X-ray data collection. This work was mixed with 3 μL of reservoir solution (50 mM Tris·HCl, 20% PEG 4000, pH supported by Russian Science Foundation Grant 18-14-00291 (to S.E.D.); by 8.6). Plates were incubated at 20 °C for 14–19 d. Crystals were stabilized by the Howard Hughes Medical Institute; and by NIH Grant GM022778 soaking for 15 min in the following buffer: 0.1 M of NaCl, 0.05 M of Tris·HCl, (to T.A.S.).

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