Calcium-dependent Self-assembly of Human Centrin 2

Abstract

Human centrin 2 (HsCen2) is a member of the EF-hand superfamily of calcium-binding proteins, often associated with the centrosomes and basal bodies. These organelles exhibit different morphological aspects, including a variety of centrin-containing fibers that connect the two centrioles or other structural elements of the pericentriolar space. The molecular basis of the Ca 2؉ -sensitive fibers and their precise role in centrosome duplication are not known. To explore the possible structural role of HsCen2, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. Using light scattering experiments, we investigated the temporal evolution of the assembly process and characterized the dependence on various chemical and physical factors, including temperature, di-cation concentration, ionic strength, protein concentration, and pH. The reversible self-assembly revealed many features of a large-size protein polymerization, with nucleation and elongation steps. Kinetic and equilibrium experiments show that a hydrophobic fluorescent probe (ANS) inhibits the polymerization by interfering with the nucleation step, probably through interactions with the apolar exposed sites on the protein surface. A truncated form of HsCen2, lacking the first 25 residues (⌬25HsCen2), shows no detectable self-assembly, pointing to the critical role played by the N-terminal fragment in the supermolecular organization of HsCen2. As revealed by isothermal titration experiments, the isolated N-terminal domains bind with a significant affinity (2 ؋ 10 5 M ؊1 ) to preformed oligomers of ⌬25HsCen2 through an entropy-driven mechanism.

The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

“…3 also allows comparison of the more distorted loops of EF-hand sites I and II in the N-terminal and without calcium present to sites III and IV. Site I differs from the more canonical HsCen-2 site IV (or EFhand I in calmodulin) mainly by the replacement of an aspartate important for Ca 2ϩ binding by Thr 45 . An analogous comparison suggests two substitutions reduce Ca 2ϩ affinity at site II, Glu 79 rather than an asparate and Asp 88 rather than a glutamate.…”

“…Titrating HsCen-2 (26,43) and other centrins (44) to Ca 2ϩ saturation is complicated by aggregation of high order oligomers, an aggregation of reversible nature. Deletion of amino acids 1-24 has little effect on protein stability, global secondary structure, or metal binding (29) but does prevent aggregation; thus, these residues must be involved in the self-assembly (45). The formation of fibers consisting of centrin may be of physiological importance in the duplication and segregation of microtubule organizing centers (21).…”

Metal-binding properties of human centrin-2 determined by micro-electrospray ionization mass spectrometry and UV spectroscopy

“…Previous studies suggested that HsCen-2 is dimeric and binds only two calciums per dimer, likely to site IV in each monomer [19]. Studies of an N-terminal 25 amino acid truncation of HsCen-2 suggest that the N-terminus is involved in the calcium-dependent dimerization of the protein [21]. On the other hand, Veeraraghavan et al showed that Chlamydomonas centrin-2 binds 4 mol of calcium/mol protein [22].…”

Biochemical and Structural Domain Analysis of Xeroderma Pigmentosum Complementation Group C Protein

“…Studies by Craescu and coworkers (72) demonstrated that the N-terminal extension of CETN2 mediated self-polymerization. To facilitate working with CETN2, a construct lacking the N-terminal 21 amino acids [CETN2 (22-172)] was generated.…”