Dynamic dimer-of-dimers architecture defines Mg²⁺ transport in human CNNM4

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• Magnesium ions (Mg2+) are vital for cellular functions.
• The precise mechanisms of Mg2+ transport across mammalian cell membranes are not fully understood.
• Mammalian CNNM proteins are implicated in Mg2+ transport, but their structures and functions remain largely elusive.

Purpose of the Study:

• To elucidate the structural basis of mammalian Mg2+ transport by human CNNM4.
• To understand the assembly, Mg2+ binding, and transport mechanism of CNNM4.
• To investigate the regulatory roles of Mg2+ and Na+ in CNNM4 activity.

Main Methods:

• Cryoelectron microscopy (cryo-EM) to determine high-resolution structures of human CNNM4.
• Biochemical assays to study Mg2+ and ATP binding.
• Functional assays to investigate Mg2+/Na+ exchange.

Main Results:

• Determined cryo-EM structures of human CNNM4 in outward-facing and occluded states.
• Revealed an unexpected tetrameric assembly (dimer of asymmetric dimers), differing from prokaryotic homologs.
• Identified Mg2+/ATP binding sites and a potential Mg2+ sensing acidic patch.
• Demonstrated Na+-dependent conformational changes in the transmembrane domain that regulate Mg2+ transport.

Conclusions:

• Established a novel mechanistic framework for mammalian CNNM-mediated Mg2+ transport.
• Highlighted significant differences between prokaryotic and eukaryotic CNNM transporters.
• Linked CNNM4 structure and function to Mg2+ homeostasis and potential human diseases.