Figure 4. Ca²⁺-binding and complex formation change the accessibility of trypsin cleavage sites in CHP3 and mCHP3.

(A) Time-dependent (0–60 min) limited proteolysis (trypsin) of CHP3 (top) and the complex of CHP3:CBD (bottom) in the presence of both Mg²⁺ and Ca²⁺. Positions of full-length protein (FL) and two major proteolytic fragments (1 and 2) as well as CBD are indicated on the right of the Coomassie-stained SDS–PAGE gel, positions of co-separated molecular mass standards (mass in kDa) – on the left; the sample containing only trypsin was loaded on the first lane (T); Figure 4—source data 1: Full gels of (A). (B) Time-dependent limited proteolysis of CHP3 and mCHP3 in the presence of Mg²⁺, Ca²⁺, both ions or in the absence of them. Sections of the gel with bands corresponding to the full-length protein (FL) and two major proteolytic fragments (1 and 2) are shown. Nearly no degradation was observed for CHP3 and mCHP3 in the complex with CBD in all conditions (Mg²⁺ + Ca²⁺ condition is presented here, other gels are shown in Figure 4—figure supplement 1B and its source data). (C) Schematic representation of full-length CHP3 with indication of N- and C-lobes, four EF-hand motifs (active EF-3 is highlighted in green). Proteolytic fragments 1 and 2 and trypsin cleavage sites were identified by mass spectrometry. (D) Combined ribbon and surface presentation of the CHP3 AlphaFold2.0 model (Varadi et al., 2022) with N- and C-lobes shown in blue and orange, respectively, and the connecting CHP-loop in cyan; the two major trypsin cleavage sites are highlighted in yellow and Ca²⁺ ion as a green sphere. The Ca²⁺ position in EF-3 was modelled by superimposition of the CHP3 model with the CHP1 X-ray structure, pdb ID 2ct9 (Andrade et al., 2004).

Figure 4—figure supplement 1. Limited trypsinolysis of CHP3 and mCHP3.

(A) Location of potential trypsin cleavage sites (Lys and Arg residues) in the CHP3 sequence. The two major sites, at which the cleavage is effective in the limited proteolysis are highlighted in red. (B) The full-size gels of limited proteolysis in different conditions. Proteins samples are indicated on the left, the ion conditions – on the top, trypsin only (T), or different CHP3 samples (CHP3, mCHP3, CHP3:CBD, or mCHP3:CBD) incubated with trypsin for 0, 5, 15, 30, 45, and 60 min were loaded from the left to the right. For a band annotation, see Figure 4A. Gels of CHP3 (MgCl₂+CaCl₂) and of CHP3:CBD (MgCl₂+CaCl₂) from Figure 4A (C) LC–ESI-TOF mass spectrometry analysis of major proteolytic fragments (1 and 2), the detected masses were 17,922 Da for the fragment 1 and 9630 Da for the fragment 2.

Figure 4—figure supplement 2. Multiple sequence alignment of human CHP1, CHP2, and CHP3.

CHP3 has one active EF-hand only, which is EF-3 (green box). EF-2 and EF-4 of CHP3 have 9-residue insertions, which disrupt the canonical Ca²⁺-coordinating loop, whereas EF-1 of CHP3 lacks the conservative negatively charged residues important for Ca²⁺ coordination (positions X, Y and –Z, respective CHP3 residues are S36, G37, and N46). EF-1, EF-2, and EF-4 that do not bind Ca²⁺ in CHP3 are shown in red rectangles. N-terminal myristoylation site is indicated with an arrow. Sequence alignment was done in BioEdit (Hall, 1999) using ClustalW algorithm.