Our group is interested in the principles underlying protein recognition by RNA, DNA, peptides and proteins, especially in the context of disease syndromes.
We have research program directed towards identification and structural characterization of small molecule inhibitors identified from experimental and in silico screens targeted to proteins and RNA scaffolds.
Messenger RNAs interact with a number of different molecules that determine the fate of each transcript and contribute to the overall pattern of gene expression. Both small molecules and proteins demonstrate a diversity of mRNA binding modes often dependent on the structural context of the regions surrounding specific target sequences. Our group remains interested in studying ligand-RNA and protein-RNA complexes that impact on human diseases.
We have written a review on the principles underlying mRNA recognition (Serganov & Patel, 2008).
Serganov, A. & Patel, D. J. (2008). Towards deciphering the principles underlying a mRNA recognition code. Curr. Opin. Struct. Biol. 18, 120-129.
RNA Endoribonucleases. A protein complex called C3PO has been shown to be a Mg2+-dependent endoRNase, which facilitates RISC activation by siRNA unwinding, as well as through removal of the cleaved passenger strand. C3PO forms a multimeric complex of translin and TRAX, in which TRAX acts as the catalytic subunit. In collaboration with the Thomas Tuschl lab (Rockefeller University), we report on the crystal structure of hexameric Drosophila C3PO formed by truncated Trax and translin, along with electron microscopic and mass spectrometric studies on full-length octameric Trax and translin (Tian et al. 2011). Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3PO’s endoribonuclease activity and interacts extensively with translin to form an octameric assembly. The catalytic pocket of Trax subunits are located within the interior chamber of the octameric scaffold.
Tian, Y. et al., Robinson, C. C., Tuschl, T. & Patel, D. J. (2011). Multimeric assembly and biochemical characterization of the Trax-translin endonuclease complex. Nat. Struct. Mol. Biol. 18, 658-664.
RNA exoribonucleases. Nibbler (Nbr) is a 3′-to-5′ exoribonuclease whose catalytic 3′-end trimming activity impacts on miRNA and piRNA biogenesis. We report on structural and functional studies in collaboration with the Stefan Ameres (IMBA-Vienna) lab to decipher the contributions of Nbr’s N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3′-end trimming (Xie et al. 2020). The NTD-core domain of A. aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding dsRNA over ssRNA. The EXO domain of A. aegypti and D. melanogaster Nbr exhibits a deep pocket lined by a DEDDy catalytic cleavage motif, that exhibits Mn2+-dependent ssRNA-specific 3′-to-5′ exoribonuclease activity. Our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.
Xie, W. et al., Brennecke, J., Ameres, S. L. & Patel, D. J. (2020). Structural and functional analysis of miRNA 3’-end trimming by Nibbler. Proc. Natl. Acad. Scis. USA. 117, 30370-30379.
RNA Decapping Enzymes. The redox cofactor NAD was discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner with Nudix hydrolase NudC was found to act as a NAD decapping enzyme in vitro and in vivo. Crystal structures of NAD decapping E. coli NudC enzyme in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product (Hofer et al. 2016). Biochemical mutation analysis in the laboratory of our collaborator Andre Jaschke (University of Heidelberg) demonstrated that the NudC is single-strand specific and needs to be homodimeric, given that the catalytic pocket is composed of amino acids from both monomers. and has a purine preference for the 5’-terminal nucleotide.
Hofer, K. et al., Patel, D. J. & Jaschke, A. (2016). Structure and function of the bacterial decapping enzyme NudC. Nat. Chem. Biol. 12, 730-734.
RNA Export. Messenger RNA export is mediated by the TAP-p15 heterodimer, which also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, thereby mediating export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical-half of CTE RNA (Teplova et a. 2011). The L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. Together with the impact of structure-guided mutations undertaken by our collaborator Elisa Izaurralde lab (Max-Planck Institute-Tubingen), our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating nuclear export of viral genomic RNAs.
Teplova, M., Wohlbold, L., Kim, N. Y., Izaurralde, E. & Patel, D. J. (2011). Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP. Nat. Struct. Mol. Biol. 18, 990-998.
Toxin-Antitoxin Regulation. MazF is an mRNA interferase, which upon activation during stress conditions, cleaves mRNAs in a sequence-specific manner, resulting in cellular growth arrest. During normal growth conditions, the MazF toxin is inactivated through binding to its cognate antitoxin, MazE. In collaboration with the Masayori Inouye lab (Rutgers University Medical School), we solved crystal structures of MazF in complex with mRNA substrate and antitoxin MazE in Bacillus subtilis (Simanshu et al. 2013). The results demonstrate that the positioning of the C-terminal helical segment of MazE within the RNA-binding channel of the MazF dimer prevents mRNA binding and cleavage by MazF.
Simanshu, D. K., Yamaguchi, Y., Park, J-H., Inouye, M. & Patel, D. J. (2013). Structural insights into mRNA recognition by toxin MazF and its regulation by antitoxin MazE in B. subtilis. Mol. Cell 52, 447-458.
Neuronal Diseases. Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. In collaboration with the Yang Shi lab (Harvard Medical School), we determined the crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, that recognize distinct trinucleotide RNA substrates (Murn et al. 2015, 2016). Structure-guided point mutations reduce the RNA-binding affinity of Unkempt both in vitro and in vivo, ablate its translational control and impair the ability of Unkempt to induce a bipolar cellular morphology, thereby highlighting its critical contribution to post-transcriptional control of neuronal morphology.
Murn, J. et al., Patel D. J., Ule, J., Luscombe, N. M., Tsai, L. H., Walsh, C. A. & Shi, Y. (2015). Control of a neural morphology program by an RNA-binding zinc finger protein, Unkempt. Genes Dev. 29, 501-512.
Murn, J., Teplova, M., Zarnack, K., Shi, Y. & Patel, D. J. (2016). Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt. Nat. Struct. Mol. Biol. 23, 16-23.
Myelination Diseases. Mammalian Quaking (QKI) is an evolutionary conserved RNA-binding protein, which post-transcriptionally regulates target genes essential for developmental processes and myelination. All QKI proteins bind RNA via their STAR domains, composed of a KH domain flanked by Qua1, critical for homodimerization, and Qua2, critical for RNA binding domains. We have solved crystal structures of the STAR domain, composed of Qua1, KH, and Qua2 motifs of QKI 1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs) (Teplova et al. 2013). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with the bound RRE. Studies on structure-guided mutations in the lab of our collaborator Thomas Tuschl (Rockefeller University) established reduced QKI RNA-binding affinity in vitro and in vivo and significantly decreased abundance of QKI target mRNAs.
Teplova, M., Hafner, M., Teplov, D., Essig, K., Tuschl, T. & Patel, D. J. (2013). Structure-function studies of STAR family Quaking proteins bound to their in vivo RNA target sites. Genes Dev. 27, 928-940.
Autoimmune Diseases. Diverse aspects of RNA metabolism are dictated by the La autoantigen, an abundant nuclear RNA-binding phosphoprotein, identified as an autoantigen in patients with systemic lupus erythematosus and Sjörgen’s syndrome. La specifically targets and protects the UUUOH 3’-terminii of nascent RNA polymerase III transcripts, while discriminating against 3’-phosphate-containing internal oligo U tracts and degraded RNA. We have solved the crystal structure of the N-terminal domain (NTD) of human La, consisting of La and RRM1 motifs, bound to a 9-mer ending in UUUOH (Teplova et al. 2006). The UUUOH 3’-end, in a splayed apart orientation, is sequestered in a basic and aromatic amino acid-lined cleft between the La and RRM1 motifs. Both hydroxyls of the sugar ring of the last U are hydrogen-bonded, with neither phosphate nor bulky modifications tolerated at this site.
Teplova, M. et al. & Patel, D. J. (2006). Structural basis for recognition and sequestration of UUUOH 3’-terminii of nascent mRNA polymerase III transcripts by La autoantigen. Mol. Cell 21, 75-85.
Muscular Dystrophy. The proteins of the muscleblind-like family, such as MBNL that harbor tandem CCCH Zn finger (ZnF) domains targeting pre-mRNAs, are important tissue-specific alternative splicing regulators that play a key role in terminal muscle differentiation. The normal splicing pattern is altered specifically in the neuromuscular disease myotonic dystrophy (DM), in part, due to inactivation of MBNL. Our crystal structure of MBNL1 ZnF3/4 bound to r(CGCUGU) established that both ZnF3 and ZnF4 specifically target GC steps with guanine and cytosine bases inserting into adjoining pockets of the protein, while the 2’-OH groups are hydrogen bonded to conserved side-chains (Teplova & Patel, 2008). The anti-parallel orientation of bound GC elements, is supportive of a chain-reversal loop trajectory for MBNL1-bound pre-mRNA targets, thereby impacting on alternative splicing regulation.
CUG binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We have solved crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements with recognition mediated by face-to-edge stacking and water-mediated hydrogen bonding networks (Teplova et al. 2010). We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation and mRNA decay.
Teplova, M. & Patel, D. J. (2008). Structural insights into RNA recognition by the alternate splicing regulator muscleblind-like MBNL1. Nat. Struct. Mol. Biol. 15, 1343-1351.
Teplova, M., Song, J., Gaw, H. Y., Teplov, V. & Patel, D. J. (2010). Structural insights into RNA recognition by the CUG binding protein 1. Structure 18, 1364-1367.
Neurodegenerative Syndromes. Nova onconeural antigens are neuron-specific RNA-binding proteins implicated in paraneoplastic opsoclonus-myoclonus-ataxia (POMA) syndrome. Nova harbors three K-homology (KH) motifs implicated in alternate splicing regulation of genes involved in inhibitory synaptic transmission. We have solved the crystal structure of the first two KH domains (KH1/2) of Nova-1 bound to an in vitro selected RNA hairpin, containing a UCAG-UCAC high-affinity binding site (Teplova et al. 2011). Sequence-specific intermolecular contacts in the complex involve KH1 and the second UCAC repeat, with the RNA scaffold buttressed by interactions between repeats. The observed anti-parallel alignment of KH1 and KH2 domains in the crystal structure of the complex, supported by binding studies in our collaborator Robert Darnell’s lab (Rockefeller University), generated a scaffold that could facilitate target pre-mRNA looping upon Nova binding, thereby potentially explaining Nova’s functional role in splicing regulation.
Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome and autism. FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with G-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. In a collaborative effort with the Alexander Serganov (New York University Medical School) and Robert Darnell labs, we report on NMR (Phan et al. 2011) and crystal structures (Vasilyev et al. 2015) of the complex between the human FMRP RGG peptide bound with an in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K(+)-stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex-quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation-π interactions, and multiple hydrogen bonds.
Phan, A. T. et al., Darnell, R. B. & Patel, D. J. (2011). Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction. Nat. Struct. Mol. Biol. 18, 796-804.
Teplova, M. et al., Darnell, R. B. & Patel, D. J. (2011). Protein-RNA and protein-protein recognition by dual KH1/2 domains of the neuronal splicing factor Nova-1. Structure 19, 930-944.
Vasilyev, N., Polonskaia, A., Darnell, J. C., Darnell, R. B., Patel, D. J. & Serganov, A. (2015). Crystal structure reveals specific recognition of a G-quadruplex RNA by a b-turn in the RGG motif of FMRP. Proc. Natl. Acad. Scis. USA. 112, E5391-E5400.
Acute Myeloid Leukemia. N6-Methyladenosine (m6A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. In a project championed by the Michaell Kharas lab (MSKCC), a genome-wide CRISPR screen identified YTHDC1 as the essential m6A reader in myeloid leukemia and that m6A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m6A condensates (nYACs) (Cheng et al. 2021). The number of nYACs increases in acute myeloid leukemia (AML) cells compared with normal hematopoietic stem and progenitor cells, with AML cells requiring the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance.
Cheng, Y. et al. Patel, D. J., Jaffrey, S. R. & Kharas, M. G. (2021). m6A mRNA catalyzes a phase-separated nuclear body that suppresses myeloid leukemic differentiation. Cancer Cell 39, 958-972.
Inhibitors Targeting Protein Scaffolds
Our group is interested in the design and structural characterization of inhibitors that target proteins mediating SARS-CoV-2 viral infection, and those involved in leukemic transformation.
Inhibitors Targeting SARS-CoV-2 Proteins. SARS-CoV-2 has a 30-kB RNA genome encoding as many as 14 open reading frames (ORFs). ORF1a and ORF1ab encode polyproteins that are processed by two essential viral proteases, 3C-like main protease (3CLpro) and papain-like protease (PLpro), which auto-excise and then cleave the polyprotein into 16 Non-structured proteins (Nsps), including the cap-0 N7-G-MTase (Nsp14) and cap1 ribose 2’-O-MTase (Nsp16). Our ongoing efforts are directed towards identifying inhibitors targeting PLpro, NSP16 and RNA-dependent RNA polymerase, as well as combination of targets.
SARS-CoV-2 RNA polymerase nsp12 shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5. In a project championed by the Jingyue Ju lab (Columbia University), it was shown that anti-HCV NS5 inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity, with cellular potencies in the mM range (Sacramento et al. 2021). Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome.
SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. In a project championed by the Jingyue Ju lab, hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir were identified as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision (Wang et al. 2022). This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.
Wang, X. et al. Patel, D. J. et al. & Ju, J. (2022). Combination of antiviral drugs to inhibit polymerase and exonuclease of SARS-C0V-2 as potential covid-19 therapeutics. Commun. Biol. 5:154.
Sacramento, C. Q. et al. Patel, D. J. et al. & Souza, T. M. (2021). The in vitro anti-viral activity of the anti-hepatitis C virus (HCV) drugs daclatasvir and sofosbuvir against SARS-CoV-2. J. Antimicrobial Chemotherapy 76, 1874-1885.
Peptide Inhibitors Targeting Leukemia Proteins. The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. In a project championed by the Michael Kharas (MSKCC), we find that the small molecule Ro 08–2750 (labeled Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays (Minuease et al. 2019). Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, together with inhibition of known MSI-targets. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model, thereby identifying a small molecule that targets MSI’s oncogenic activity.
Minuesa, G. et al. & Patel, D. J., Goldgur, Y., Chodera, J. D. & Kharas, M. G. (2019). Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia. Nat. Commun. 10:2691.
Our group has undertaken structure-functional investigation of helicases, motor-nucleases, DSB repair proteins and transcriptional repressors that function at DNA complex level.
Mycobacterial Helicases. Our collaborator Stewart Shuman’s lab (MSKCC) has shown that Mycobacterium smegmatis Lhr (MsmLhr), which contains an N-terminal helicase domain with a distinctive tertiary structure (Lhr-Core) and a C-terminal domain (Lhr-CTD) of unknown structure, is the founder of a novel clade of bacterial helicases that exhibit ATPase, translocase and ATP-dependent helicase activities. Lhr translocates 3’ to 5’ on ssDNA and unwinds secondary structures enroute, with RNA:DNA hybrid being preferred versus DNA:DNA duplex. We employed cryo-EM to solve the structure of the CTD of full-length MsmLhr, showing how that the CTD adopts a unique homo-tetrameric quaternary structure, with each protomer composed of a series of five-winged helix (WH) modules and a b-barrel module (Warren et al. 2021).
Warren, G. M., Wang, J., Patel, D. J. & Shuman, S. (2021). Oligomeric quarternary structure of Escherichia coli and Mycobacterium smegmatis Lhr helicases is nucleated by a novel C-terminal domain composed of five winged-helix modules. Nucleic Acids Res. 49, 3876-3887.
Mycobacterial Motor-Nucleases. Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. In collaboration with the Stewart Shuman lab, we solved the cryo-EM structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5′ single-strand DNA (ssDNA) tail by the AdnA nuclease (Jia et al. 2019). The structures reveal the path of the 5′ ssDNA through the AdnA nuclease domain and the mechanism of 5′ strand cleavage; the path of the 3′ tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5′ and 3′ strands. The research was extended to AdnAB mutants wherein a key Trp was replaced with other amino acids with retention of activity in ssDNA-dependent ATP hydrolysis, but displayed a gradient of effects on DSB resection (Warren et al. 2022). Based on a cryo-EM structure of the Trp to Ala mutant, we conclude that the nucleobase-stacking tryptophan is critical for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB.
Jia, N., Unciuleac, M. C., Xue, C., Greene, E. C., Patel, D. J. & Shuman, S. (2019). Structure and single-molecule kinetic analysis of the mycobacterial motor-nuclease AdnAB illuminate the mechanism of DNA double-strand break resection. Proc. Natl. Acad. Scis. USA. 116, 24507-24516.
Warren, G. M., Meier, A., Wang, J., Patel, D. J., Greene, E. C. & Shuman, S. (2022). Structure-activity relations at a nucleobase-stacking tryptophan required for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB. Nucleic Acids Res., 50, 952-961.
Shieldin Complex in DNA Repair. The Shieldin complex, composed of REV7, SHLD1, SHLD2 and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA+ ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. We solved the crystal structure of fused human SHLD2–SHLD3-REV7 ternary complex thereby revealing that Shieldin assembly requires induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7 (Xie et al. 2021). We also report the cryo-EM structure of ATPgS-bound fused human SHLD2-SHLD3-REV7-TRIP13 complex, thereby demonstrating that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel.
Xie, W., Wang, S., Wang, J., De la Cruz, J., Xu, G., Scaltriti, M. & Patel, D. J. (2021). Molecular mechanisms of assembly and remodeling of human Shieldin complex. Proc. Natl. Acad. Scis. USA. 118, e2024512118.
BEN Domain Transcriptional Repressors. The BEN domain family of transcriptional repressors are a recently recognized DNA-binding module that is present in diverse metazoans and certain viruses. In collaboration with the Eric Lai (MSKCC) and Aiming Ren (Zhejiang University) labs, we applied detailed functional, genomic, and structural studies on several BEN domain (BD) factors from both Drosophila and humans, towards an explanation of their preference for distinct DNA binding sites (Dai et al. 2013, 2015, Zheng et al. 2022) .
Dai, Q., Ren, A., Westholm, J. O., Serganov, A., Patel, D. J. & Lai, E. C. (2013). The BEN domain is a novel sequence-specific DNA binding domain conserved in neural transcriptional repressors. Genes Dev. 27, 602-614.
Dai, Q., Ren, A., Westholm J. O., Patel, D. J. and Lai, E. (2015). Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family. Genes Dev. 29, 48-62.
Zheng, L. et al., Patel, D. J., Zhang, L., Prasanth, S., Yu, Y, Ren, A. & Lai, E. C. (2022). Distinct structural bases for sequence-specific DNA binding by mammalian BEN domains. Genes Dev. 36, 225-240.
Sexual Differentiation in Malaria Parasites. Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary to infect the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. In a project championed by the Bjorn Kafsack lab (Weill-Cornell Medical School), a recently identified Homedomain Protein 1 (HDP1) was shown to be a DNA-binding protein first expressed in gametocytes, that enhances the expression of key genes critical for early sexual differentiation (Campello-Norillo et al. 2022).
Campello-Morillo, R. A. et al. Patel, D. J., Nobel, W. S., Llinas, M., Le Roch, K. G. & Kafsack, B. F. (2022). The transcriptional regulator HDP1 controls expansion of the inner membrane complex during early sexual differentiation of malaria parasites. Nat. Microbiol. 7, 288-299.
Our group has collaborated with colleagues to solve peptide-protein and protein-protein structures, which when combined with functional studies, provide mechanistic insights into the biological systems of interest.
AML1-ETO Contribution to Leukemogenesis. Oligomerization of fusion protein AML1-ETO, mediated by its NHR2 domain, is critical for leukemogenesis. The lab of our collaborator Robert Roeder lab (Rockefeller University) showed that AML1-ETO functions through a stable AML1-ETO-containing transcription factor complex (AEFTC) that contains several haematopoietic transcription co-factors, including E proteins that contain a novel NHR2-binding (N2B) motif. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which the N2B peptide makes direct contact with a dimeric alignment of NHR2 domains, providing a novel model of how transcription factors create a new protein-binding interface through dimerization/oligomerization (Sun et al. 3013).
Sun, X-J. et al., Patel, D. J., Nimer, S. D. & Roeder, R. G. (2013). A stable transcription factor complex nucleated by dimeric AML1-ETO controls leukemogenesis. Nature 500, 93-97.
WSTF regulates the DNA damage response. DNA double-stranded breaks (DSBs) present a serious challenge for eukaryotic cells, with inability to repair breaks leading to genomic instability, carcinogenesis and cell death. During the DSB response, mammalian chromatin undergoes reorganization demarcated by H2A.X Ser139 phosphorylation. In a collaborative project championed by the David Allis lab (Rockefeller University), a new regulatory mechanism has been identified that is mediated by WSTF (Williams-Beuren syndrome transcription factor), a component of the WSTF-ISWI ATP-dependent chromatin-remodeling complex (Xiao et al. 2009). The results show that WSTF phosphorylates Tyr142 of H2A.X, and that WSTF activity has an important role in regulating several events that are critical for the DNA damage response.
Xiao, A. et al. Patel, D. J., Elledge, S. J. & Allis, C. D. (2009). WSTF regulates the DNA damage response of H2A.X via a novel tyrosine kinase activity. Nature 457, 57-62.
Paf1 Complex in Transcriptional Regulation. The polymerase-associated factor 1 (Paf1) complex is a general transcription elongation factor of RNA polymerase II, which is composed of five core subunits, Paf1, Ctr9, Cdc73, Leo1 and Rtf1, that broadly affects gene expression. In a collaboration championed by the Zhanxin Wang lab (Beijing Normal University, Beijing), the crystal structure was solved of the core region composed of the Ctr9-Paf1-Cdc73 ternary complex from thermophilic fungi Deng et al. 2018). We find that Ctr9 is composed of 21 tetratricopeptide repeat (TPR) motifs that wrap three circular turns in a right-handed superhelical manner around the N-terminal region of an elongated single polypeptide-chain scaffold of Paf1. The Cdc73 fragment is positioned within the surface groove of Ctr9, where it contacts mainly with Ctr9 and minimally with Paf1. Our work provides structural insights into the molecular mechanisms of the Paf1 complex in transcriptional regulation.
Deng, P. et al. Roeder, R. G., Patel, D. J. & Wang, Z. (2018). Transcriptional elongation factor Paf1 core complex adopts a spirally-wrapped solenoidal topology. Proc. Natl. Acad. Scis. USA. 115, 9998-10003.
Polycomb-like proteins in PRC2 Recruitment. The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression and has essential roles in maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, are PRC2-associated factors that form subcomplexes with PRC2 core components to modulate PRC2’s enzymatic activity. In a collaboration championed by the Zhanxin Wang lab, crystal structures were solved of the N-terminal domains of PHF1 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides (Li et al. 2017). We show that the extended homologous region folds into a winged-helix structure, which specifically binds with a unique alignment to the unmethylated CpG motif.
Li, H. et al. Patel, D. J., Bulyk, M., Shi, Y. and Wang, Z. (2017). Polycomb-like proteins link the PRC2 complex with CpG islands. Nature 549, 287-291.
Chromatin-Remodeling Factor SMARCA3. p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin reuptake inhibitors (SSRIs). In collaboration with the Paul Greengard lab (Rockefeller University), we show that SMARCA3, a chromatin-remodeling factor, is a target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer, with complex formation increasing the DNA-binding affinity of SMARCA3 and its localization to the nuclear matrix fraction (Oh et al. 2013).
Oh, Y-S. et al., Patel, D. J., Kim, Y. & Greengard, P. (2013). SMARCA3, a chromatin remodeling factor, is required for p11-dependent anti-depressive action. Cell 152, 831-843.
Inhibitors Targeting RNA Scaffolds
Our group is interested in the molecular basis for site-specific aminoglycoside recognition both on natural and in vitro selected RNA targets. Several groups have identified RNA folds following in vitro selection that target aminoglycoside antibiotics with affinities ranging from mM to nM. These RNA aptamer sequences are likely to undergo adaptive binding on complex formation to generate specific pockets for the bound aminoglycoside antibiotics.
Aminoglycoside Antibiotics Targeting RNA. Tobramycin is an aminoglycoside antibiotic used to treat cystic fibrosis-associated bacterial, lower respiratory tract and urinary tract infections. We have solved the solution structure of the aminoglycoside antibiotic tobramycin complexed with a stem-loop RNA aptamer whereby the RNA aptamer ’zippers up’ alongside the attached stem through mismatch formation (Jiang & Patel, 1998). The tobramycin inserts into the deep groove centered about the mismatch pairs and is partially encapsulated between its floor and a looped-out guanine base that flaps over the bound antibiotic.
The aminocyclitol antibiotic streptomycin interacts with the central domain of 16S ribosomal RNA and also inhibits group I intron splicing. A modular streptomycin-binding RNA aptamer with mM affinity has been identified by in vitro selection We solved the crystal structure of the complex between streptomycin and an in vitro selected RNA aptamer (Tereshko et al. 2003). The RNA aptamer, which contains two asymmetric internal loops, adopts a distinct cation-stabilized fold involving a series of S-shaped backbone turns anchored by canonical and non-canonical pairs and triples. The streptomycin streptose ring is encapsulated by stacked arrays of bases from both loops at the elbow of the L-shaped RNA architecture.
Apramycin is unique among aminoglycoside antibiotics in containing a bicyclic core domain, whereby it binds preferentially to eukaryotic decoding sites and induces misreading of the genetic code during translation. The solved crystal structure of the complex demonstrates binding of the apramycin in a deep groove of the decoding site RNA, which forms a continuously stacked helix comprising novel non-canonical pairs and a bulged adenine with interactions mediated by a Mg cation and water molecules. (Hermann et al. 2007).
Jiang, L. & Patel, D. J. (1998). Solution structure of the tobramycin-RNA aptamer complex. Nature Struct. Biol. 5, 769-774.
Tereshko, V., Skripkin, E. & Patel, D. J. (2003). Encapsulating streptomycin within a small 40-mer RNA. Chem. Biol. 10, 175-187.
Hermann, T., Tereshko, V., Skripkin, E. & Patel, D. J. (2007). The structure of the apramycin-eukaryotic RNA decoding site complex. Blood Cells, Molecules, and Diseases 38, 193-198.