Trop2 Forms a Stable Dimer with Significant Structural Differences within the Membrane-Distal Region as Compared to EpCAM

Abstract

Trop2 is a cell-surface transmembrane glycoprotein involved in the maintenance of epithelial tissue integrity and is an important carcinoma marker. It shares similar claudin-interaction capacity with its paralogue EpCAM, and both are implicated in signaling triggered by proteolytic cleavage within the ectodomain. However, the cell proliferation-regulating interactions with IGF-1, neuregulin-1, and α5β1 integrin appear to be Trop2-specific. To illuminate the structural differences between Trop2 and EpCAM, we report the first crystal structure of a Trop2 ectodomain dimer and compare it to the analogous part of EpCAM. While the overall fold of the two proteins is similar, the dimers differ. In Trop2, the inter-subunit contacts are more extensive than in EpCAM, and there are two major differences in the membrane-distal regions. The immunogenic N-terminal domain is in Trop2 almost colinear with the dimer interface plain and consequently more laterally exposed, and the cleft of yet unknown functionality between the two subunits is almost absent. Furthermore, the site of initial signaling-associated proteolytic cleavage in Trop2 is accessible in the dimeric state, while in EpCAM dimer destabilization is required. The structural differences highlight the divergent evolutionary path of the two proteins and pave the way for their structure-based utilization in therapy.

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).…”

“…For the MD simulations, we used with minor modifications the protocol described by Modenutti et al 66 We utilized as initial structure a model built from the crystal structures of the cis‐dimer of the extracellular domain of human Trop‐2 (PDB IDs 7PEE 44 and 7E5N 45 ) including only eight residues from the transmembrane region.…”

“…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 We then examined the likely consequences of GDLD-associated missense mutations by mapping them onto the Trop-2 homology model. GDLD-associated mutations C108R and C119S are likely to cause severe misfolding accompanied by severe loss of function, as both Cys residues are involved in a disulfide bond in the extracellular domain.…”

An assembly of TROP2-mediated signaling events

“…This demonstrated increased nuclear TROP2 ICD and attenuated NUMB expression results into reduced recurrence‐free survival probability in human prostate cancer (Ju et al 2016). Trerotola et al (2021) reported that ADAM Metallopeptidase Domain 10 (ADAM10) also cleaves TROP2 between the arginine and threonine residues (R87 and T88), which triggers the progression of malignant tumors of skin, ovary, colon, lung, and pancreas (Pavsic 2021; Trerotola et al 2021). The study in human colorectal and pancreatic cancer cell lines reported that TROP2 phosphorylation (S322) by Protein Kinase C α / δ has a causal effect on the interaction with claudin‐7 (CLDN7), leading to its mislocalization, which results into the loss of membrane‐localized CLDN7, which enhances cell motility (Mori et al 2019).…”

An EpCAM/Trop2 mechanostat differentially regulates collective behaviour of human carcinoma cells

“…All properties reported for EpCAM appear to be shared by Trop2, including oligomerization, association with tetraspanin-based domains, with claudins, potential interaction with the cytoskeleton, or matriptase-mediated cleavage (Balzar et al, 2001(Balzar et al, , 1998Fagotto and Aslemarz, 2020;Guerra et al, 2022;Kuhn et al, 2007;Nubel et al, 2009;Pavsic et al, 2014;Pavšič, 2021;Szabo et al, 2022;Wu et al, 2020). Both form homodimers, but structural considerations indicate incompatibility for heterodimers (Pavšič, 2021).…”