The Catalytic Copper of Peptidylglycine α-Hydroxylating Monooxygenase also Plays a Critical Structural Role

Abstract

Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.

Not All Secretory Granules Are Created Equal: Partitioning of Soluble Content Proteins

“…However, the mGFP and DsRed sequences are only ϳ30% identical. Because PHMcc molecules differing at a single amino acid crystallized under different conditions (Siebert et al, 2005), PHM-GFP and PHM-DsRed would not be expected to cocrystallize in immature granules. Consis- Figure 8.…”

Metals in Alzheimer’s Disease: A Combined Experimental and Numerical Approach

“…Experimental data suggest that the four involved His's are made by the two pairs of consecutive, His 13 and His 14 , residues coming from two adjacent A -peptides. This structure gives rise to a very special arrangement, as structural databases report one single instance where a metal (actually Cu) is bound to two His's that are one next to the other along the protein sequence [96]. There are instead many cases where Cu is coordinated to four His's side-chains belonging to the same monomer, but all of them concern proteins belonging to the wide class of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) proteins.…”

Characterization of the basic charge variants of a human IgG1

“…21 In addition, the copper also plays an important roles in the structure and molecular trafficking of the PAM. 22,23 However, exactly how pro-amidation is mediated by certain ions remains unclear.…”

“…To determine the nature of the basic peaks that appeared sensitive to copper concentration, three separate productions were conducted with different copper concentrations to supply material for characterization. A pH-IEC method 22 was used to isolate the basic charge variants; the pH-IEC profile is shown in Figure 3A. The three major basic peaks (B1-B3 in Fig.…”