The solution structure of the MANEC-type domain from hepatocyte growth factor activator inhibitor-1 reveals an unexpected PAN/apple domain-type fold

Abstract

A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins.

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

“…(8). The location of the MANEC is as reported (22). The internal domain sequences correspond to that determined in this study.…”

“…Due to the inherent instability of the proposed internal domain, we decided to investigate it in the context of its neighboring domains. The MANEC-internal domain construct was unsuccessful in producing detectable amounts of protein in yeast cultures, although a similar approach had successfully produced the isolated MANEC (22). However, a construct covering the internal domain and Kunitz-1 (IK1) expressed well in yeast and the purified protein eluted from repeated size exclusion chromatography experiments as a stable monomer (Fig.…”

“…Based on our recent structure of the MANEC domain (22) and the two existing structures of Kunitz-1 in complex with HGFA and matriptase (40,41), the sequence gap between the MANEC and Kunitz-1 (Arg 153 -Tyr 249 ) was analyzed by the more advanced sequencebased homology search server, I-TASSER (Fig. 5B).…”

“…MANEC, internal domain, the LDLR class A domain, and Kunitz-2) are all important for the regulation of the inhibitory activity of HAI-1, most likely by participating in intramolecular interdomain interactions resulting in different tertiary structures with corresponding varying inhibitory activities (17). As a first step to provide structural evidence of how the non-inhibitory domains of HAI-1 may contribute to the overall function of the protein, we recently completed a structural characterization of the N-terminal MANEC domain, defining it as a new subclass of the PAN/apple family of domains with numerous examples of regulatory properties as mediators of inter-and intramolecular protein-protein interactions (22).…”

Applications of high dimensionality experiments to biomolecular NMR

“…Similary, the determination of the global fold of the 82 kDa monomeric enzyme malate synthase G necessitated the use of a 4D methyl-methyl NOESY experiment [178]. Also, more recently 4D NUS NOESY spectra were employed to obtain a high quality structure of MANEC domain [179].…”