Structural insights into the cis and trans assembly of human trophoblast cell surface antigen 2

Abstract

Summary Human trophoblast cell surface antigen 2 (TROP-2) is an important target of tumor therapy, and antibody-drug conjugates with sacituzumab targeting TROP-2 have been approved for the treatment of triple-negative breast cancer. Here, we report the crystal structures of TROP-2-ECD, which can be either cis- or trans- dimers depending on which distinct but overlapping interfaces is used to engage with monomers. The cis- or trans -tetrameric forms of TROP-2 can also be assembled with a non-overlapping interface with either cis - or trans- dimerization, suggesting that cis - and trans- dimers cluster on the cell surface. The binding site of sacituzumab on TROP-2 is mapped to be located on a stretched polypeptide in CPD (Q237-Q252), which is not involved in either cis- or trans- interactions. The present findings will improve understanding of the molecular assembly of TROP-2 on tumor cells and shed light on future design of biologics for tumor therapy.

hIMB1636-MMAE, a Novel TROP2-Targeting Antibody-Drug Conjugate Exerting Potent Antitumor Efficacy in Pancreatic Cancer

“…hIMB1636, developed by our laboratory, is a novel humanized antibody against TROP2 . Although it had a similar K D value to sacituzumab, hIMB1636 had advantages over sacituzumab in the following aspects: binding to the conformational epitope of TROP2 protein, not a linear epitope like sacituzumab; inducing more potent ADCC; and showing more significant antitumor effects both in vitro and in vivo . Additionally, we had previously demonstrated that 64 Cu/ 177 Lu-labeled hIMB1636 was effective for the diagnosis and treatment of pancreatic cancer, and TROP2 could be a potential therapeutic target for pancreatic cancer …”

Rescue of secretion of rare‐disease‐associated misfolded mutant glycoproteins in UGGT1 knock‐out mammalian cells

“…In order to evaluate the likely impact of each mutation, we started by building a homology model of the dimer of the mature portion of human Trop‐2 (Uniprot P09758, TACD2_HUMAN). Besides the AlphaFoldMultimer model, 42 a model was also built using Modeller 43 : the extracellular portion of the dimer (residues 27–268) was generated using as templates two distinct crystal structures of the cis‐dimer of the same region of human Trop‐2 (PDB IDs 7PEE 44 and 7E5N 45 ); the transmembrane region (residues 278–298) was based on the NMR‐derived model of the transmembrane portion of the rat p75 protein (PDB ID 4MZV, 30% sequence identity over 21 residues); the cytoplasmic domain of human Trop‐2 (residues 299–323) was based on the NMR structure of the C‐term of Trop‐2 (PDB ID 2MAE).…”

“…Histidine protonation was assigned by favoring for each residue the formation of hydrogen bonds. All disulfide bonds as reported by Sun et al 45 were enforced/preserved throughout. The complete protonated systems were then solvated by a truncated cubic box of OPC waters, ensuring that the distance between the biomolecule surface and the box limit was at least 10 Å.…”

“…This area of the protein is distal to the membrane and it is involved in the trans dimer-of-dimers that establishes cell-cell adhesions. 45 The charge-reversal mutation E227K sits close to the dimer interface, surrounded by three positively charged residues: K72, K231 and R228: this is likely to destabilize the monomer and/or cause loss of protein dimerisation. The L186P mutation affects a small surface pocket not too far from the membrane.…”

Assessing PDB Macromolecular Crystal Structure Confidence at the Individual Amino Acid Residue Level

“…The obverse scenario of high pLDDT versus low RSCC was also examined for representative cases. PDB: 7E5M (Sun et al, 2021) is MX structure of residues 33-268 of human tumor-associated calcium signal transducer 2 determined at 3.2 Å resolution (UniProt ID P09758), with median RSCC∼0.67 versus median pLDDT∼94 for the corresponding AlphaFoldDB CSM. The experimental structure and the CSM are very similar, with C α RMSD∼0.5 Å when loop residues 82-103 are excluded from the 3D comparison (Supplementary Figure S6).…”