Structural Basis For Recognition Research Articles

Structural basis for recognition and methylation of p97 by METTL21D, a valosin-containing protein lysine methyltransferase

p97 is a human AAA+ (ATPase associated with diverse cellular activities, also known as valosin-containing protein [VCP]) ATPase, which is involved in diverse cellular processes such as membrane fusion and proteolysis. Lysine-specific methyltransferase of p97 (METTL21D) was identified as a class I methyltransferase that catalyzes the trimethylation of Lys315 of p97, a so-called VCP lysine methyltransferase (VCPKMT). Interestingly, VCPKMT disassembles a single hexamer ring consisting of p97-D1 domain and methylates Lys315 residue. Herein, the structures of S-adenosyl-L-methionine-bound VCPKMT and S-adenosyl-L-homocysteine-bound VCPKMT in complex with p97 N/D1 (N21-Q458) were reported at a resolution of 1.8Å and 2.8Å, respectively. The structures revealed the molecular details for the recognition and methylation of monomeric p97 by VCPKMT. Using biochemical analysis, we also investigated whether the methylation of full-length p97 could be sufficiently enhanced through cooperation between VCPKMT and the C terminus of alveolar soft part sarcoma locus (ASPL). Our study provides the groundwork for future structural and mechanistic studies of p97 and inhibitors.

Structural basis for recognition and regulation of arenavirus polymerase L by Z protein

Junin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections.

Abstract 1836: Structural basis for recognition of MUC16 expressed in various cancers

Abstract Background: Overexpressed Mucin 16 (MUC16) is highly related with cancer progression, metastasis, and therapy resistance in multiple malignancies. The proteolytic cleavages of MUC16 form independent bimodular fragments, a circulating shed form known as an ovarian cancer biomarker (CA 125) and a C-terminal tail-connected juxtamembrane region as an unshed form which is critical in determining the unique mechanistic roles of MUC16 and therapeutic interventions. Effective neutralizing antibody for MUC16-positive solid cancers is urgently needed for earlier diagnosis and immunotherapeutic approaches. Methods: The novel 4H11-scFv (single chain antibody) targeting MUC16-juxtamembrane was optimized for mammalian cell usage and Liter-scale cultures of HEK293T were infected with high-titer lentiviral stocks expressing the 4H11-scFv and the recombinant proteins were purified by a series of purification process and prepared for further characterization and crystallization. The sequence corresponding to human MUC16-juxtamembrane was cloned with maltose binding protein (MBP) and expressed by the bacterial system. The purified MBP-fused-MUC16 was prepared for co-crystallization with 4H11-scFv. A series of biochemical studies are performed: Thermal stability measurement of 4H11-scFv, MBP-pull down, Isothermal titration calorimetry (ITC) for interaction analysis, bioactivity of Alexa-labeled 4H11-scFv, analytical size-exclusion chromatography, multi-mutagenesis studies at the interface of 4H11-scFv-MUC16 complex. Crystallizations were performed using high-throughput crystallization screen kits, followed by extensive manual optimization. X-ray diffraction data were collected at the Advanced Photon Source (APS) and processed with HKL2000, PHENIX, CCP4 suite package and COOT. Results: We determined the crystal structures of 4H11-scFv at 2.36 Å and 4H11-scFv-MUC16 at 2.47 Å resolutions, respectively. Structural analysis of a 4H11-scFv demonstrated that all six complementarity-determining regions (CDRs) are composed of 5 loop-like coiled coil regions and 1 short-helix in VH (Heavy chain), while juxtamembrane portion showing flexible loop regions with β-Turns. The complex was stabilized through extensive hydrogen bond-mediated interaction, salt bridge, and cation-π interaction. We identified some specific residues of MUC16, critical to form the complex, from mutagenesis studies. Biochemical analysis demonstrated the 4H11-scFv potently neutralizes MUC16 and stable at the cancer microenvironmental and endosomal pH with a possibility of pre-clinical diagnosis/treatment in various cancers. Conclusion: We investigate the structures and the neutralizing mechanisms for 4H11-scFv targeting the MUC16. Our study offer insight into antibody to neutralize the non-cleaved cell surface retained portion of MUC16 and advance our ability to design anti-MUC16 antibody with improved therapeutic properties. Citation Format: Kwangkook Lee, Kay Perry, Oladapo O. Yeku, David R. Spriggs. Structural basis for recognition of MUC16 expressed in various cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1836.

Structural basis for recognition and ring-cleavage of the Pseudomonas quinolone signal (PQS) by AqdC, a mycobacterial dioxygenase of the α/β-hydrolase fold family

The cofactor-less dioxygenase AqdC of Mycobacteroides abscessus catalyzes the cleavage and thus inactivation of the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone), which plays a central role in the regulation of virulence factor production by Pseudomonas aeruginosa. We present here the crystal structures of AqdC in its native state and in complex with the PQS cleavage product N-octanoylanthranilic acid, and of mutant AqdC proteins in complex with PQS. AqdC possesses an α/β-hydrolase fold core domain with additional helices forming a cap domain. The protein is traversed by a bipartite tunnel, with a funnel-like entry section leading to an elliptical substrate cavity where PQS positioning is mediated by a combination of hydrophobic interactions and hydrogen bonds, with the substrate’s C4 carbonyl and C3 hydroxyl groups tethered by His97 and the catalytic His246, respectively. The side chain of the AqdC-bound product extends deeper into the “alkyl tail section” of the tunnel than PQS, tentatively suggesting product exit via this part of the tunnel. AqdC prefers PQS over congeners with shorter alkyl substituents at C2. Kinetic data confirmed the strict requirement of the active-site base His246 for catalysis, and suggested that evolution of the canonical nucleophile/His/Asp catalytic triad of the hydrolases to an Ala/His/Asp triad is favorable for catalyzing dioxygenolytic PQS ring cleavage.

Structural basis for recognition and repair of the 3'-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis.

NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3′-phosphate and other 3′-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3′-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3′-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3′-phosphatase NExo bound to DNA in the presence and absence of a 3′-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3′-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1.

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex.

116. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

116. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

相関構造生物学によるUHRF1のヒストン認識機構の解明

相関構造生物学によるUHRF1のヒストン認識機構の解明

Structural basis for recognition of the type VI spike protein VgrG3 by a cognate immunity protein.

The bacterial type VI secretion system (T6SS) is used by donor cells to inject toxic effectors into receptor cells. The donor cells produce the corresponding immunity proteins to protect themselves against the effector proteins, thereby preventing their self-intoxication. Recently, the C-terminal domain of VgrG3 was identified as a T6SS effector. Information on the molecular mechanism of VgrG3 and its immunity protein TsaB has been lacking. Here, we determined the crystal structures of native TsaB and the VgrG3C-TsaB complex. VgrG3C adopts a canonical phage-T4-lysozyme-like fold. TsaB interacts with VgrG3C through molecular mimicry, and inserts into the VgrG3C pocket.

The structural basis for recognition and modification of the 30S ribosomal subunit by an antibiotic resistance methyltransferase (569.3)

Ribosomal RNA contains modifications at specific nucleotides in all organisms, some of which control resistance to antibiotics. However, the mechanisms are not well understood controlling how modification enzymes recognize their specific target nucleotide and position it within the active site. We report the crystal structure of the pathogen‐derived aminoglycoside‐resistance rRNA methyltransferase NpmA bound to the 30S ribosomal subunit in a pre‐catalytic state. The structure reveals that NpmA recognizes conserved rRNA tertiary structure rather than RNA sequence. Additionally, the structure shows NpmA flips the target nucleotide from its position within the helical base stack to position it within the active site. The structure provides a general framework for investigating the mechanisms of other rRNA modification enzymes.Grant Funding Source: Supported by National Institutes of Health

DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.

Crystal Structures of Two Archaeal 8-Oxoguanine DNA Glycosylases Provide Structural Insight into Guanine/8-Oxoguanine Distinction

Among the four DNA bases, guanine is particularly vulnerable to oxidative damage and the most common oxidative product, 7,8-dihydro-8-oxoguanine (8-oxoG), is the most prevalent lesion observed in DNA molecules. Fortunately, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Ogg enzymes are divided into three separate families, namely, Ogg1, Ogg2, and archaeal GO glycosylase (AGOG). To date, structures of members of both Ogg1 and AGOG families are known but no structural information is available for members of Ogg2. Here we describe the first crystal structures of two archaeal Ogg2: Methanocaldococcus janischii Ogg and Sulfolobus solfataricus Ogg. A structural comparison with OGG1 and AGOG suggested that the C-terminal lysine of Ogg2 may play a key role in discriminating between guanine and 8-oxoG. This prediction was substantiated by measuring the glycosylase/lyase activity of a C-terminal deletion mutant of MjaOgg.

Chaperone-Assisted Crystallography with DARPins

The structure of proteins that are difficult to crystallize can often be solved by forming a noncovalent complex with a helper protein--a crystallization "chaperone." Although several such applications have been described to date, their handling usually is still very laborious. A valuable addition to the present repertoire of binding proteins is the recently developed designed ankyrin repeat protein (DARPin) technology. DARPins are built based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein. The broad potential of these binding proteins for X-ray crystallography is illustrated by five cocrystal structures that have been determined recently comprising target proteins from distinct families, namely a sugar binding protein, two kinases, a caspase, and a membrane protein. This article reviews the opportunities of this technology for structural biology and the structural aspects of the DARPin-protein complexes.

Structural Basis for Recognition and Sequestration of UUU OH 3′ Temini of Nascent RNA Polymerase III Transcripts by La, a Rheumatic Disease Autoantigen

The nuclear phosphoprotein La was identified as an autoantigen in patients with systemic lupus erythematosus and Sjogren's syndrome. La binds to and protects the UUU(OH) 3' terminii of nascent RNA polymerase III transcripts from exonuclease digestion. We report the 1.85 angstroms crystal structure of the N-terminal domain of human La, consisting of La and RRM1 motifs, bound to r(U1-G2-C3-U4-G5-U6-U7-U8-U9OH). The U7-U8-U9OH 3' end, in a splayed-apart orientation, is sequestered within a basic and aromatic amino acid-lined cleft between the La and RRM1 motifs. The specificity-determining U8 residue bridges both motifs, in part through unprecedented targeting of the beta sheet edge, rather than the anticipated face, of the RRM1 motif. Our structural observations, supported by mutation studies of both La and RNA components, illustrate the principles behind RNA sequestration by a rheumatic disease autoantigen, whereby the UUU(OH) 3' ends of nascent RNA transcripts are protected during downstream processing and maturation events.

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta.

X-ray analysis of enzyme-DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9-10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (DeltaG degrees approximately -8.7 to -9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1-DNA interactions provide only one order of magnitude (DeltaG degrees approximately -1.1 to -1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.

Structural Basis for Recognition and Catalysis by the Bifunctional dCTP Deaminase and dUTPase from Methanococcus jannaschii

Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure–function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42Å and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77Å and 2.10Å, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase β barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.

Structural basis for recognition by an in vitro evolved affibody.

The broad binding repertoire of antibodies has permitted their use in a wide range of applications. However, some uses of antibodies are precluded due to limitations in the efficiency of antibody generation. In vitro evolved binding proteins, selected from combinatorial libraries generated around various alternative structural scaffolds, are promising alternatives to antibodies. We have solved the crystal structure of a complex of an all alpha-helical in vitro selected binding protein (affibody) bound to protein Z, an IgG Fc-binding domain derived from staphylococcal protein A. The structure of the complex reveals an extended and complementary binding surface with similar properties to protein-antibody interactions. The surface region of protein Z recognized by the affibody is strikingly similar to the one used for IgG(1) Fc binding, suggesting that this surface contains potential hot-spots for binding. The implications of the selected affibody binding-mode for its application as a universal binding protein are discussed.

Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA.

Spontaneous oxidation of guanine residues in DNA generates 8-oxoguanine (oxoG). By mispairing with adenine during replication, oxoG gives rise to a G x C --> T x A transversion, a frequent somatic mutation in human cancers. The dedicated repair pathway for oxoG centres on 8-oxoguanine DNA glycosylase (hOGG1), an enzyme that recognizes oxoG x C base pairs, catalysing expulsion of the oxoG and cleavage of the DNA backbone. Here we report the X-ray structure of the catalytic core of hOGG1 bound to oxoG x C-containing DNA at 2.1 A resolution. The structure reveals the mechanistic basis for the recognition and catalytic excision of DNA damage by hOGG1 and by other members of the enzyme superfamily to which it belongs. The structure also provides a rationale for the biochemical effects of inactivating mutations and polymorphisms in hOGG1. One known mutation, R154H, converts hOGG1 to a promutator by relaxing the specificity of the enzyme for the base opposite oxoG.

High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein.

Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain.

Crystallization of RNA-protein complexes

The widespread occurrence of RNA-protein interactions in critical cellular processes makes an understanding of the structural basis for recognition and sequence discrimination of paramount importance. Until recently, however, the difficulty in obtaining large quantities of homogeneous RNA had made structural investigations of many interesting species by biophysical methods impossible. Summarized here are the recent applications of several in vivo expression systems to the overproduction of a number of RNA species. The potential usefulness in crystallization of RNA synthesized in vitro by T7 RNA polymerase is also discussed. Together with advancements in separation methods by HPLC, these two methods make a much wider range of systems accessible to structural analysis. A comparison of the successful cocrystallizations of two complexes of amino-acyl-tRNA synthetases with their cognate tRNA substrates is presented.