Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea–Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

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mSphere of Influence: Structural Insights into the Molecular Mechanism Underlying Placental Malaria

mSphere ◽

10.1128/msphere.00391-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Maria del Pilar Quintana

Keyword(s):

Plasmodium Falciparum ◽

Chondroitin Sulfate ◽

Placental Malaria ◽

Structural Basis ◽

Content Type ◽

Link Type ◽

Structural Details ◽

Inhibitory Antibodies ◽

Structural Insights ◽

Insight Into

ABSTRACT Maria del Pilar Quintana works on immunology and pathogenesis of severe malaria. In this mSphere of Influence article, she reflects on how the papers “Structural basis for placental malaria mediated by Plasmodium falciparum VAR2CSA” (R. Ma, T. Lian, R. Huang, J. P. Renn, J. D. Petersen, J. Zimmerberg, P. E. Duffy, N. H. Tolia, Nat Microbiol 6:380–391, 2021, https://doi.org/10.1038/s41564-020-00858-9) and “Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding” (K. Wang, R. Dagil, T. Lavsten, S. K. Misra, C. B. Spliid, Y. Wang, T. Gustavsson, D. R. Sandoval, E. E. Vidal-Calvo, S. Choudary, M. O. Agerback, K. Lindorff-Larsen, M. A. Nielsen, T. G. Theander, J. S. Sharp, T. M. Clausen, P. Gourdon, A. Salanti [Research Square preprint], 2021, https://doi.org/10.21203/rs.3.rs-121821/v1) shed light on the precise structural details behind Plasmodium falciparum VAR2CSA binding to chondroitin sulfate A (CSA) in the placenta and how these novel insights have changed the way she will approach her work toward the discovery of new broadly cross-reactive/inhibitory antibodies targeting VAR2CSA.

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Cryo-EM structures of undocked innexin-6 hemichannels in phospholipids

Science Advances ◽

10.1126/sciadv.aax3157 ◽

2020 ◽

Vol 6 (7) ◽

pp. eaax3157 ◽

Cited By ~ 3

Author(s):

Batuujin Burendei ◽

Ruriko Shinozaki ◽

Masakatsu Watanabe ◽

Tohru Terada ◽

Kazutoshi Tani ◽

...

Keyword(s):

Gap Junctions ◽

Structural Basis ◽

Wild Type ◽

Amino Terminal ◽

Functional Assays ◽

Cryo Electron Microscopy ◽

Dynamics Simulations ◽

Open States ◽

Insight Into ◽

Junction Proteins

Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo–electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.

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Computational insight into the mechanisms of action and selectivity of Afraxis PAK inhibitors

Future Medicinal Chemistry ◽

10.4155/fmc-2019-0273 ◽

2020 ◽

Vol 12 (5) ◽

pp. 367-385

Author(s):

Di Han ◽

Huiqun Wang ◽

Wei Cui ◽

Beibei Zhang ◽

Bo-Zhen Chen

Keyword(s):

Molecular Dynamics ◽

Mechanisms Of Action ◽

Structural Basis ◽

Free Energies ◽

Activity Data ◽

Experimental Activity ◽

Inhibitory Mechanisms ◽

Dynamics Simulations ◽

Insight Into ◽

Binding Free Energies

Aim: The p21-activated kinases (PAKs) are involved in many important biological activity regulations. FRAX019, FRAX414, FRAX597, FRAX1036 and G-5555 were identified as PAKs inhibitors. Their detailed inhibitory mechanisms deserve further investigation. Results: Molecular dynamics simulations and further calculations for the PAK1/inhibitor and PAK4/inhibitor complexes indicate that their binding free energies are basically consistent with the trend of experimental activity data. Conclusion: The anchoring of residues Leu347PAK1 and Leu398PAK4 is the structural basis for designing Afraxis PAK inhibitors. This study discloses the inhibitory mechanisms of FRAX019, FRAX414, FRAX597, FRAX1036 and G-5555 toward PAK1 and PAK4 and some clues to enhance kinase activities and selectivities, which will provide valuable information to the development of more potent and selective PAK inhibitors.

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Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

10.1101/2020.06.02.131110 ◽

2020 ◽

Author(s):

Iga Kucharska ◽

Elaine Thai ◽

Ananya Srivastava ◽

John Rubinstein ◽

Régis Pomès ◽

...

Keyword(s):

Malaria Vaccine ◽

Humoral Response ◽

Circumsporozoite Protein ◽

Inhibitory Antibody ◽

Structural Ordering ◽

X Ray ◽

X Ray Crystallography ◽

Antibody Recognition ◽

Structural Details ◽

Dynamics Simulations

ABSTRACTPlasmodium sporozoites express circumsporozoite protein (CSP) on their surface, an essential protein that contains central repeating motifs. Antibodies targeting this region can neutralize infection, and the partial efficacy of RTS,S/AS01 – the leading malaria vaccine against P. falciparum (Pf) – has been associated with the humoral response against the repeats. Although structural details of antibody recognition of PfCSP have recently emerged, the molecular basis of antibody-mediated inhibition of other Plasmodium species via CSP binding remains unclear. Here, we analyze the structure and molecular interactions of potent monoclonal antibody (mAb) 3D11 binding to P. berghei CSP (PbCSP) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We reveal that mAb 3D11 can accommodate all subtle variances of the PbCSP repeating motifs, and, upon binding, induces structural ordering of PbCSP through homotypic interactions. Together, our findings uncover common mechanisms of antibody evolution in mammals against the CSP repeats of Plasmodium sporozoites.

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C-Glycoside metabolism in the gut and in nature: Identification, characterization, structural analyses and distribution of C-C bond-cleaving enzymes

Nature Communications ◽

10.1038/s41467-021-26585-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Takahiro Mori ◽

Takuto Kumano ◽

Haibing He ◽

Satomi Watanabe ◽

Miki Senda ◽

...

Keyword(s):

Bond Cleavage ◽

Structural Basis ◽

Electron Microscopic ◽

Intestinal Microbes ◽

X Ray ◽

Sugar Moiety ◽

Structural Details ◽

Catalytic Mechanisms ◽

Cleavage Reactions ◽

Biochemical Analyses

AbstractC-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.

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Structural ordering of the Plasmodium berghei circumsporozoite protein repeats by inhibitory antibody 3D11

eLife ◽

10.7554/elife.59018 ◽

2020 ◽

Vol 9 ◽

Author(s):

Iga Kucharska ◽

Elaine Thai ◽

Ananya Srivastava ◽

John L Rubinstein ◽

Régis Pomès ◽

...

Keyword(s):

Plasmodium Species ◽

Humoral Response ◽

Circumsporozoite Protein ◽

Inhibitory Antibody ◽

Structural Ordering ◽

X Ray ◽

X Ray Crystallography ◽

Antibody Recognition ◽

Structural Details ◽

Dynamics Simulations

Plasmodium sporozoites express circumsporozoite protein (CSP) on their surface, an essential protein that contains central repeating motifs. Antibodies targeting this region can neutralize infection, and the partial efficacy of RTS,S/AS01 – the leading malaria vaccine against P. falciparum (Pf) – has been associated with the humoral response against the repeats. Although structural details of antibody recognition of PfCSP have recently emerged, the molecular basis of antibody-mediated inhibition of other Plasmodium species via CSP binding remains unclear. Here, we analyze the structure and molecular interactions of potent monoclonal antibody (mAb) 3D11 binding to P. berghei CSP (PbCSP) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We reveal that mAb 3D11 can accommodate all subtle variances of the PbCSP repeating motifs, and, upon binding, induces structural ordering of PbCSP through homotypic interactions. Together, our findings uncover common mechanisms of antibody evolution in mammals against the CSP repeats of Plasmodium sporozoites.

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Structural basis for carbapenam C-alkylation by TokK, a B12-dependent radical SAM enzyme

10.1101/2021.07.10.451909 ◽

2021 ◽

Author(s):

Hayley Knox ◽

Erica Sinner ◽

Craig Townsend ◽

Amie Boal ◽

Squire Booker

Keyword(s):

Functional Class ◽

Essential Medicines ◽

Structural Basis ◽

Antibiotic Biosynthesis ◽

Radical Mechanism ◽

Radical Sam ◽

X Ray ◽

Radical Sam Enzyme ◽

Carbapenem Antibiotic ◽

Insight Into

Cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes acting during carbapenem antibiotic biosynthesis carry out radical-mediated methyl transfers that underlie the therapeutic usefulness of these essential medicines. Here we present x-ray crystal structures of TokK, which are representative of this functional class, containing its two metallocofactors and determined in the presence and absence of carbapenam substrate. The structures give the first visualization of a cobalamin-dependent radical SAM methylase that employs the radical mechanism shared by a vast majority of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggests that substrate positioning governs the rate of each methylation event.

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RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114065119 ◽

2022 ◽

Vol 119 (3) ◽

pp. e2114065119

Author(s):

Juntaek Oh ◽

Tiezheng Jia ◽

Jun Xu ◽

Jenny Chong ◽

Peter B. Dervan ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Minor Groove ◽

Dna Lesions ◽

Structural Basis ◽

Rna Pol Ii ◽

Pol Ii ◽

X Ray ◽

A Minor ◽

Insight Into

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

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A different mechanism of C-type inactivation in the Kv-like KcsA mutant E71V

10.1101/2021.09.22.461404 ◽

2021 ◽

Author(s):

Ahmed Rohaim ◽

Bram J.A. Vermeulen ◽

Jing Li ◽

Felix Kümmerer ◽

Federico Napoli ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Bound Water ◽

Selectivity Filter ◽

Structural Basis ◽

Water Molecules ◽

X Ray ◽

Physiological Importance ◽

Kv Channels ◽

Dynamics Simulations

ABSTRACTA large class of K+ channels display a time-dependent phenomenon called C-type inactivation whereby prolonged activation by an external stimulus leads to a non-conductive conformation of the selectivity filter. C-type inactivation is of great physiological importance particularly in voltage-activated K+ channels (Kv), affecting the firing patterns of neurons and shaping cardiac action potentials. While understanding the molecular basis of inactivation has a direct impact on human health, its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the pH-activated bacterial K+ channel KcsA, whose selectivity filter under inactivating conditions adopts a constricted conformation at the level of the central glycine (TTVGYGD) that is stabilized by tightly bound water molecules. However, C-type inactivation is highly sensitive to the molecular environment surrounding the selectivity filter in the pore domain, which is different in Kv channels than in the model KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is consistently substituted by a nonpolar valine in most Kv channels, suggesting that this side chain is an important molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and molecular dynamics simulations of the E71V mutant of KcsA is undertaken to explore the features associated with this Kv-like construct. In both X-ray and ssNMR data, it is observed that the filter of the Kv-like KcsA mutant does not adopt the familiar constricted conformation under inactivating conditions. Rather, the filter appears to adopt a conformation that is slightly narrowed and rigidified over its entire length. No structural inactivation water molecules are present. On the other hand, molecular dynamics simulations indicate that the familiar constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like E71V mutation in the KcsA channel may be associated with different modes of C-type inactivation, showing that distinct selectivity filter environments entail distinct C-type inactivation mechanisms.

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