Modulation of calmodulin plasticity by the effect of macromolecular crowding - PubMed (original) (raw)

Modulation of calmodulin plasticity by the effect of macromolecular crowding

Dirar Homouz et al. J Mol Biol. 2009.

Abstract

In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells, which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we used the well-characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell-shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca(2+), leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions, in which crowding effects enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff, analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca(2+) and target proteins.

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Figures

Figure 1

(a) A schematic representation of a coarse-grained CaM in the presence of Ficoll 70. CaM is represented by a side-chain Cα model. The N-lobe of CaM is shown in blue, while the C-lobe is shown in red. Ficoll is displayed as hard-spheres with a radius of 55 Å, shown in gold. (b) A ribbon representation of all-atom CaM (PDB: 1CFD) with attached fluorophores. The color code of the lobes is the same as in (a). Alexa488 (in green) is the donor fluorophore used in the FRET studies and is shown attached to position 34 while QSY9 (in magenta) is the quencher fluorophore and is shown attached to position 110. CaM is 148 amino acid residues in length.

Figure 2

Computer simulation of the distribution of the donor-acceptor distance, r_DA, of CaM in the bulk case (black) and in the presence of 40% volume fraction of Ficoll70 (red) at 0.96_Tf.

Figure 3

(a) Emission spectra of CaMDA in the absence (0% w/v) and presence of Ficoll 70 (40 % w/v). Inset shows the normalized fluorescence of donor-alone labeled CaM. (b) _r_DA was calculated for CaMDA in the various buffer conditions (−Ca2+, +Ca2+ and with Ca2+ and target peptide) from emission spectra similar to the one shown in panel (a) as described in Materials and Methods. On the same bar plot the separation distance, _r_DA, shows a decrease in distance from Ficoll 70 at 20% (w/v) to 40% (w/v).

Figure 4

Free energy diagram as a function of the structural overlap function of the N-lobe of CaM (χN) and the shape parameter (Δ) at T=0.96 Tf (a) in the bulk system and (b) in the presence of crowding agents at φc=40%. The color bar is scaled by kBT, where kB is the Boltzmann constant. The white area represents where the value of free energy exceeds 8.5 kBT. Typical ensemble structures pointing to circled basins are reconstructed to all-atomistic protein models using the SCAAL program The Ca2+-binding segments of CaM are: EF1 in blue, EF2 in cyan, EF3 in yellow and EF4 in red.

Figure 5

Free energy profiles as a function of the structural overlap function of the C-lobe of CaM (χC) and the shape parameter (Δ) at T=0.96 Tf (a) in the bulk system and (b) in the presence of crowding agents at φc=40%. The color bar is scaled by kBT, where kB is the Boltzmann constant. The white area represents where the value of free energy exceeds 8.5 kBT.

Figure 6

The distribution of the angles, ρ(cos(θ)), formed by adjacent helices in the individual EF hands of CaM. This distribution is shown for EF hands in the two lobes of CaM as well as three different collapsed states (ND, NC, and CC). (a) A schematic diagram shows the angle formed by adjacent helices in each EF hand. (b) ρ(cos(θ))of two helices (A/B) in EF 1 and (C/D) in EF2 in the N- lobe. (c) ρ(cos(θ)) of two helices (E/F) in EF 3 and (G/H) in EF4 in the C-lobe.

Figure 7

The distribution of the angles, ρ(cos(θ)) formed by helices in different EF hands of CaM. This distribution is shown for EF hands in the two lobes of CaM as well as the three different collapsed states (ND, NC, and CC). (a) A schematic diagram shows the angle formed by helices of different EF hands within one lobe of CaM. (b) ρ(cos(θ)) of two helices (A/D) in the N-lobe and (E/H) in the C-lobe.

Figure 8

Schematic representation of changes in the free energy profiles for the N- and C-lobes of CaM in the absence and presence of crowding agents. In buffer, the ND state is the dominant one in which the two lobes preserve their native contacts. The other less populated states (NC and CC) are more spherical than the ND state. However, they have different distribution of the native contacts. In both NC and CC states, the C-lobe has lost most of its native contacts while the N-lobe is more native like in the NC state and becomes less native like in the CC state. In the presences of crowding agents, an increase in free energy of the system increases the probability that transitions will occur between the different states. Because the N-lobe is less flexible, the relative proportion of molecules in each state remains similar. In contrast, the increased flexibility of the C-lobe causes a change in the distribution of the states with ND being less populated in favor of the NC and CC states as crowding increases the free energy. We propose that the less flexible nature of the N-lobe produces more subtle changes in its structure as the free energy of the system increases so that it behaves more like a rheostat. In contrast, as the free energy of the system increases the C-lobe behaves more like a switch as it transitions more abruptly from native to collapses states.

References

1. Laurent TC, Ogston AG. The interaction between polysaccharides and other macromolecules. 4. the osmotic pressure of mixtures of serum albumin and hyaluronic acid. Biochem. J. 1963;89:249–253. - PMC - PubMed
1. Berg OG. The influence of macromolecular crowding on thermodynamic activity: solubility and dimerization constants for spherical and dumbbell-shaped molecules in a hard-sphere mixture. Biopolymers. 1990;30:1027–1037. - PubMed
1. Minton AP. Models for excluded volume interaction between an unfolded protein and rigid macromolecular cosolutes: Macromolecular crowding and protein stability revisited. Biophys J. 2005;88:971–985. - PMC - PubMed
1. Reiss H, Frisch H, Lebowitz JL. Statistical mechanics of rigid spheres. J. Chem. Phys. 1959;31:369–380.
1. Cheung MS, Klimov D, Thirumalai D. Molecular crowding enhances native state stability and refolding rates. Proc Natl Acad Sci U S A. 2005;102:4753–4758. - PMC - PubMed

Modulation of calmodulin plasticity by the effect of macromolecular crowding - PubMed (original) (raw)