Simultaneous Binding of Cu⁺ and Cu²⁺ at the Two-Tiered Copper Binding Site of the Intestinal Mucin MUC2

Mucin glycoproteins are secreted from epithelial goblet cells to create protective barriers lining the intestines, stomach, lungs, and other body surfaces. MUC2 is the primary glycoprotein secreted in the intestine and is essential for intestinal homeostasis. The D1 segment of the MUC2 N-terminal region was recently shown to bind Cu²⁺ and Cu⁺ separately in a unique two-tiered binding site. Copper is an essential metal acquired through diet for cells and enzymes to function properly, but little is known about how it is handled in the digestive tract. With both oxidation states of Cu in the intestine, we asked how the binding of Cu⁺ to MUC2 D1 impacts the binding of Cu²⁺ and vice versa. Here, we use a combination of competition titrations, electron paramagnetic spectroscopy, and X-ray absorption spectroscopy to characterize the physical properties of Cu²⁺ and Cu⁺ binding to MUC2 D1 at pH values relevant to the intestine. Our data show that simultaneous yet noncooperative binding of Cu²⁺ and Cu⁺ is possible and further reveal new insights into the pH dependence and plasticity of the Cu²⁺ and Cu⁺ binding sites. These results inspire interesting questions about the functional roles of MUC2 Cu handling in the intestinal tract.