Molecular basis for the broad substrate selectivity of a peptide prenyltransferase

The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.

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The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902211116 ◽

2019 ◽

Vol 116 (41) ◽

pp. 20404-20410 ◽

Cited By ~ 7

Author(s):

Amy J. Fernandez ◽

Earnest James Paul Daniel ◽

Sai Pooja Mahajan ◽

Jeffrey J. Gray ◽

Thomas A. Gerken ◽

...

Keyword(s):

Colorectal Cancer ◽

Molecular Basis ◽

Catalytic Domain ◽

Binding Pocket ◽

Substrate Selectivity ◽

Peptide Substrate ◽

X Ray ◽

Biochemical Studies ◽

Cancer Mutations ◽

Insight Into

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

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Structural basis for peptide substrate specificities of glycosyltransferase GalNAc-T2

10.1101/2020.06.25.171371 ◽

2020 ◽

Author(s):

Sai Pooja Mahajan ◽

Yashes Srinivasan ◽

Jason W. Labonte ◽

Matthew P. DeLisa ◽

Jeffrey J. Gray

Keyword(s):

False Positive Rate ◽

Structural Basis ◽

Amino Acid Residues ◽

Peptide Substrate ◽

Structural Motifs ◽

Peptide Substrates ◽

Substrate Preferences ◽

Wide Range ◽

Peptide Interactions ◽

Positive Rate

AbstractThe polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isozymes in humans—an unusually large number—unique to O-glycosylation. GalNAc-Ts exhibit both redundancy and finely tuned specificity for a wide range of peptide substrates. In this work, we deciphered the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform. Our approach involved sampling and characterization of peptide–enzyme conformations obtained from Rosetta Monte Carlo-minimization–based flexible docking. We computationally scanned 19 amino acid residues at positions −1 and +1 of an eight-residue peptide substrate, which comprised a dataset of 361 (19×19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and non-glycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5% and a false positive rate of 7.3%. The glycosylatable peptide substrates viz. peptides with proline, serine, threonine, and alanine at the −1 position of the peptide preferentially exhibited cognate sequon-like conformations. The preference for specific residues at the −1 position of the peptide was regulated by enzyme residues R362, K363, Q364, H365 and W331, which modulate the pocket size and specific enzyme-peptide interactions. For the +1 position of the peptide, enzyme residues K281 and K363 formed gating interactions with aromatics and glutamines at the +1 position of the peptide, leading to modes of peptide-binding sub-optimal for catalysis. Overall, our work revealed enzyme features that lead to the finely tuned specificity observed for a broad range of peptide substrates for the GalNAc-T2 enzyme. We anticipate that the key sequence and structural motifs can be extended to analyze specificities of other isoforms of the GalNAc-T family and can be used to guide design of variants with tailored specificity.

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Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

10.1101/2021.05.22.445248 ◽

2021 ◽

Author(s):

Saikat Bhattacharya ◽

Suman Wang ◽

Divya Reddy ◽

Siyuan Shen ◽

Ying Zhang ◽

...

Keyword(s):

Protein Interactions ◽

Molecular Basis ◽

Structural Basis ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Related Protein ◽

Protein Protein Interactions ◽

Recognition Motif ◽

Splicing Regulators ◽

Hnrnp Proteins

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

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Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

Nature Communications ◽

10.1038/s41467-021-26799-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Saikat Bhattacharya ◽

Suman Wang ◽

Divya Reddy ◽

Siyuan Shen ◽

Ying Zhang ◽

...

Keyword(s):

Protein Interactions ◽

Molecular Basis ◽

Structural Basis ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Related Protein ◽

Protein Protein Interactions ◽

Recognition Motif ◽

Splicing Regulators ◽

Hnrnp Proteins

AbstractThe RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

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Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2012.674249 ◽

2012 ◽

Vol 30 (1) ◽

pp. 62-73 ◽

Cited By ~ 2

Author(s):

Valeriy G. Ostapchenko ◽

Marine E. Gasparian ◽

Yurij A. Kosinsky ◽

Roman G. Efremov ◽

Dmitry A. Dolgikh ◽

...

Keyword(s):

Catalytic Subunit ◽

Structural Basis ◽

Substrate Selectivity

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Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

Biochemical Journal ◽

10.1042/bcj20170328 ◽

2017 ◽

Vol 474 (20) ◽

pp. 3373-3389 ◽

Cited By ~ 9

Author(s):

Dong-Dong Meng ◽

Xi Liu ◽

Sheng Dong ◽

Ye-Fei Wang ◽

Xiao-Qing Ma ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Glycoside Hydrolase ◽

Complex Structure ◽

Dynamics Simulation ◽

Structural Basis ◽

Glycoside Hydrolase Family ◽

Substrate Selectivity ◽

Glucose Residue ◽

Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

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Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

eLife ◽

10.7554/elife.05553 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 22

Author(s):

Harriet Lane-Serff ◽

Paula MacGregor ◽

Edward D Lowe ◽

Mark Carrington ◽

Matthew K Higgins

Keyword(s):

Innate Immunity ◽

Trypanosoma Brucei ◽

Molecular Basis ◽

Structural Basis ◽

Β Subunit ◽

Trypanosome Infection ◽

Lateral Mobility ◽

African Trypanosomes ◽

Distal Half ◽

Immunity Factor

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

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The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome

eLife ◽

10.7554/elife.16381 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 4

Author(s):

Kathleen L McCann ◽

Takamasa Teramoto ◽

Jun Zhang ◽

Traci M Tanaka Hall ◽

Susan J Baserga

Keyword(s):

Protein Interactions ◽

Molecular Basis ◽

Large Subunit ◽

Ribosomal Subunit ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Rrna Processing ◽

Protein Protein Interactions ◽

Recognition Motif ◽

Processing Defects

ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4’s hub function by abrogating several of Nop4’s protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease.

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Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

10.1101/2021.03.17.435767 ◽

2021 ◽

Author(s):

Hauke S. Hillen ◽

Elena Lavdovskaia ◽

Franziska Nadler ◽

Elisa Hanitsch ◽

Andreas Linden ◽

...

Keyword(s):

Molecular Basis ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Large Subunit ◽

Structural Basis ◽

Catalytic Core ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

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Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920097117 ◽

2020 ◽

Vol 117 (20) ◽

pp. 10806-10817 ◽

Cited By ~ 6

Author(s):

Michael P. Torrens-Spence ◽

Ying-Chih Chiang ◽

Tyler Smith ◽

Maria A. Vicent ◽

Yi Wang ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Convergent Evolution ◽

Structural Features ◽

Divergent Evolution ◽

Structural Basis ◽

Acid Decarboxylase ◽

Substrate Selectivity ◽

Amino Acid Decarboxylase ◽

Catalytic Mechanisms

Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

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