Dynamic regulation of Zn(II) sequestration by calgranulin C

Abstract

Calgranulin C performs antimicrobial activity in the human immune response by sequestering Zn(II). This biological function is afforded with the aid of two structurally distinct Ca(II)-binding EF hand motifs, wherein one of which bears an unusual amino acid sequence. Here, we utilize solution state NMR relaxation measurements to investigate the mechanism of Ca(II)-modulated enhancement of Zn(II) sequestration by calgranulin C. Using C¹³ /N¹⁵ CPMG dispersion experiments we have measured pH-dependent major and minor state populations exchanging on micro-to-millisecond timescale. This conformational exchange takes place exclusively in the Ca(II)-bound state and can be mapped to residues located in the EF-I loop and the linker between the tandem EF hands. Molecular dynamics (MD) simulations spanning nano-to-microsecond timescale offer insights into the role of pH-dependent electrostatic interactions in EF-hand dynamics. Our results suggest a pH-regulated dynamic equilibrium of conformations that explore a range of "closed" and partially "open" sidechain configurations within the Zn(II) binding site. We propose a novel mechanism by which Ca(II) binding to a non-canonical EF loop regulates its flexibility and tunes the antimicrobial activity of calgranulin C.

Keywords: Ca(II) binding proteins; NMR spectroscopy; molecular dynamics simulations; zinc sequestration.

FIGURE 1

(a) Metal‐binding scaffold of calgranulin C (PDB: 1ODB). Cu(II) and Zn(II) share the His₃Asp‐binding site, which is occupied by Zn(II) under biological conditions and (b) sequence comparison of residues of EF‐I and ‐II loops in calgranulin A, B, and C

FIGURE 2

Residue‐specific R _ex contributions to ¹⁵N R ₂ rates from CPMG relaxation dispersion experiments at 11.7 T (hollow circles) and 18.8 T (filled circles) for (a) apo and, (b) Ca(II)‐calgranulin C at pH 6.0 and 25°C. The secondary structure of the protein is displayed at the top of the plots: α‐helices (blue bars); β‐sheet (yellow arrow); loop (solid line). (c, d, e) R _ex values mapped on the crystal structure of Ca(II)‐calgranulin C (PDB code: 1GQM) with (d) showing only the EF‐I and II loops and (e) showing the hinge region. Gray spheres represent Ca(II). Region 1 includes residues 12–34 and 68, 69; region 2 includes residues 39–47

FIGURE 3

Plot of logarithm of forward (k _a) and backward (k _b) rates from individual residue fits showing two separate exchange processes (dashed circles)

FIGURE 4

¹⁵N^H‐CPMG relaxation dispersion curves of representative residues from region 1 at pH 6.0 (blue) and 5.5 (yellow) acquired at 11.7 (hollow circles) and 18.8 T (filled circles) and 25°C. (a) Reliable fits could be obtained due to large fluctuations in data points; (b) resonance was not observed at 18.8 T

FIGURE 5

Residue‐specific R _ex contributions to ¹⁵N R ₂ at 11.7 T (hollow circles) and 18.8 T (filled circles) for Ca(II)‐calgranulin C at pH 5.5. R _ex values were determined as described in Figure 2

FIGURE 6

Van't Hoff plots showing temperature dependences of exchange processes in regions 1 and 2 with the calculated thermodynamics parameters in the inset

FIGURE 7

¹³C^ε1 CPMG relaxation dispersion trajectories for histidines in Ca(II)‐calgranulin C at pH 6.0 and 25°C. Experiments were performed at 11.7 T (hollow circles) and 18.8 T (filled circles). Solid lines represent fits to the trajectories using parameters provided in the text

FIGURE 8

Crystal structures of (a) apo (PDB: 2WCF) and (b) Ca(II)‐protein (PDB: 1E8A) showing the interaction between the two EF loops with the backbones of β‐strand forming residues shown as sticks. (c) Sidechain fluctuations in the His₃Asp motif for all 12 chains in the crystal structure of Ca(II)‐protein (PDB: 1GQM)

FIGURE 9

(a) Ca(II)‐calgranulin C; residues shown in red comprise the hinge region and residues in the blue monomer are referred to as X'. (b) Root mean square fluctuations. Frequency of hydrogen bond/salt bridge interactions for (c) H87, (d) D25. (e) Frequency of D25‐H85′ salt bridge and (f) average D25 C^α–H85' C^α distance for each simulation replicate. (g) Porcupine plot and (h) distribution of simulation conformers along PC1. (i) Porcupine plot and (j) distribution of simulation conformers along PC2. Porcupine plot arrows indicate the directions of fluctuations, and arrows <2 Å in length are omitted for visual clarity. In PC1, increasing values along the coordinate correlate with pressing of V68 and D69 of EF‐II toward the EF‐I loop. In PC2, increasing values along the coordinate correlate with pushing D25 away from H85′