Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells

Area of Science:

• Biochemistry
• Structural Biology
• Extracellular Matrix Research

Background:

• Microfibril-associated glycoprotein 4 (MFAP4) is an extracellular matrix glycoprotein implicated in organ fibrosis, COPD, and cardiovascular diseases.
• MFAP4's roles in aortic aneurysms and its interactions with elastogenic proteins (fibrillin-1, tropoelastin) and cells (via integrins) are established, but its structural details remain elusive.

Purpose of the Study:

• To determine the three-dimensional structure of human MFAP4 using cryo-electron microscopy.
• To elucidate the molecular interfaces responsible for MFAP4's interactions with other proteins and cellular components.
• To investigate the role of calcium and disulfide bonds in MFAP4 assembly and function.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to obtain high-resolution structural data of human MFAP4.
• Atomic model building into the cryo-EM map to define structural features and interaction interfaces.
• Biochemical binding assays to assess interactions with fibrillin-1, tropoelastin, LTBP4, and small fibulins.
• Site-directed mutagenesis (C34S) to investigate the role of disulfide bonds in assembly and function.

Main Results:

• The cryo-EM structure reveals that MFAP4 assembles into an octamer in the presence of calcium, composed of two sets of homodimers.
• Homodimers are stabilized by intermolecular disulfide bonds, and octamer assembly involves salt-bridge and non-polar interactions.
• MFAP4 exhibits multiple protein-protein interaction surfaces, largely dependent on its octameric assembly, and these interactions are largely unaffected by a C34S mutation.
• MFAP4-fibrillin-1 assemblies were found to inhibit MFAP4's interaction with cells.

Conclusions:

• The determined structure of the MFAP4 octamer provides critical insights into its assembly mechanism and interaction interfaces.
• MFAP4's multimeric state is crucial for its diverse protein-protein interactions, influencing its biological roles in extracellular matrix organization and cell signaling.
• Understanding MFAP4 structure-function relationships, particularly its interaction with fibrillin-1, may offer therapeutic targets for fibrotic and cardiovascular diseases.