Crystal Structures of Human C4.4A Reveal the Unique Association of Ly6/uPAR/α-neurotoxin Domain

Abstract

Ly6/uPAR/α-neurotoxin domain (LU-domain) is characterized by the presence of 4-5 disulfide bonds and three flexible loops that extend from a core stacked by several conversed disulfide bonds (thus also named three-fingered protein domain). This highly structurally stable protein domain is typically a protein-binder at extracellular space. Most LU proteins contain only single LU-domain as represented by Ly6 proteins in immunology and α-neurotoxins in snake venom. For Ly6 proteins, many are expressed in specific cell lineages and in differentiation stages, and are used as markers. In this study, we report the crystal structures of the two LU-domains of human C4.4A alone and its complex with a Fab fragment of a monoclonal anti-C4.4A antibody. Interestingly, both structures showed that C4.4A forms a very compact globule with two LU-domain packed face to face. This is in contrast to the flexible nature of most LU-domain-containing proteins in mammals. The Fab combining site of C4.4A involves both LU-domains, and appears to be the binding site for AGR2, a reported ligand of C4.4A. This work reports the first structure that contain two LU-domains and provides insights on how LU-domains fold into a compact protein and interacts with ligands.

Crystal structure of TEX101, a glycoprotein essential for male fertility, reveals the presence of tandemly arranged Ly6/uPAR domains

“…Most TFPD proteins contain a single TFPD, while LYPD3 and uPAR have two and three TFPDs, respectively [27,33]. TEX101 also has two TFPDs, as revealed in the current study.…”

“…sequence identity 22%; Fig. S2A) [27]. We found that TEX101 indeed contained two LU domains (LU1 and LU2), both with a three-fingered protein domain (TFPD) fold (Fig.…”

“…The TFPD fold was first identified in snake neurotoxins, since this time, at least 45 TFPD proteins have been identified in the human genome, including proteins of the Ly6/uPAR family and receptors of the TGF-b superfamily [35,36]. The crystal structures of some TFPD proteins in complex with their interacting partners have been reported, including receptors of the TGF-b superfamily with their ligand proteins (PDB code: 3KFD) [30], CD59 with vaginolysin (PDB code: 5IMY) [31], and GPIHBP1 and lipoprotein lipase (PDB code: 6E7K) [32], LYPD3 with a Fab fragment of mouse monoclonal antibody 11H10 raised against LYPD3 (PDB code: 6ION) [27], and uPAR with uPA (PDB code: 2FD6) [33] (Fig. 5A).…”

“…The structural epitope of LYPD3 recognized by the Fab fragment is primarily located on the back, and edge, of the LU1 hand (b4, b6, b7), while the loop region connects b10 and b11 of LU2 (LYPD3 structural element numbers are assigned to correspond to those of TEX101; Fig. 5A) [27]. This region is also predicted to be involved in interactions with AGR2, a reported physiological ligand for LYPD3, via docking calculation [27].…”

“…S3). We compared the LU domain structures of TEX101 with the TFPD domains of other TFPD proteins, namely TGF-bR1 (PDB code: 3KFD) [30], CD59 (PDB code: 5IMY) [31], GPIHBP1 (PDB code: 6E7K) [32], human LYPD3 (PDB code: 6IOM) [27], and human uPAR (PDB code: 2FD6) [33] (Fig. S3).…”