Crystal structures of the vitamin D-binding protein and its complex with actin: structural basis of the actin-scavenger system

Abstract

Actin is the most abundant protein in eukaryotic cells, but its release from cells into blood vessels can be lethal, being associated with clinical situations including hepatic necrosis and septic shock. A homeostatic mechanism, termed the actin-scavenger system, is responsible for the depolymerization and removal of actin from the circulation. During the first phase of this mechanism, gelsolin severs the actin filaments. In the second phase, the vitamin D-binding protein (DBP) traps the actin monomers, which accelerates their clearance. We have determined the crystal structures of DBP by itself and complexed with actin to 2.1 A resolution. Similar to its homologue serum albumin, DBP consists of three related domains. Yet, in DBP a strikingly different organization of the domains gives rise to a large actin-binding cavity. After complex formation the three domains of DBP move slightly to "clamp" onto actin subdomain 3 and to a lesser extent subdomain 1. Contacts between actin and DBP throughout their extensive 3,454-A(2) intermolecular interface involve a mixture of hydrophobic, electrostatic, and solvent-mediated interactions. The area of actin covered by DBP within the complex approximately equals the sum of those covered by gelsolin and profilin. Moreover, certain interactions of DBP with actin mirror those observed in the actin-gelsolin complex, which may explain how DBP can compete effectively with gelsolin for actin binding. Formation of the strong actin-DBP complex proceeds with limited conformational changes to both proteins, demonstrating how DBP has evolved to become an effective actin-scavenger protein.

Figure 1

Structure of uncomplexed DBP. (a and b) Two ribbon representations of the DBP structure are shown rotated by 90°. The three domains of DBP are colored burgundy (domain I), blue (domain II), and orange (domain III). Notice how the α-helix X of domain I (best seen in b) and α-helix VIII of domain II (best seen in a) are bent. The structural changes occurring within these two α-helices, which connect domains I and II and II and III of DBP, respectively, account for an entirely different domain organization in DBP as compared with HSA. To better illustrate this point, superimpositions of the two α-helices with their HSA counterparts are shown in c and d (where the corresponding regions of HSA are shown in gray). The arrows in c and d show how the differences between the two proteins can be separated into motions in two nearly perpendicular directions. Note, however, that when analyzed separately the domains of DBP and albumin clearly are related. Thus, whereas domain III of DBP is significantly shorter than that of HSA, domains I and II of the two proteins are related more closely (rms deviations of 2.4 and 2.08 Å for 150 and 145 equivalent Cα positions, respectively).

Figure 2

Structure of the actin–DBP complex. (a) Stereo view of the structure of the complex. The three domains of DBP are colored as described for Fig. 1, and actin is colored green. Gly-227 and Thr-414 of DBP constitute the first amino acids of domains II and III, respectively. (b) Sequence of DBP. The amino acids from the three domains are colored as described for a. Superimposed on the sequence is a representation of the secondary structure assignment. Regions of DBP that interact with actin are underlined in black. Under these underlined sequences of DBP we show the interacting segments in the primary structure of actin (green). (c) Stereo representation of a characteristic section of the 2F_o − F_c electron density map (contoured at 1.5σ) at the interface between actin (yellow trace) and DBP (gray trace). Notice how numerous well defined water molecules mediate many of the interactions between actin and DBP.

Figure 3

Common interactions of gelsolin and DBP with actin. The α-helical segments Ser-194–Lys-207 of DBP and Gln-71–Leu-84 of gelsolin domain 1 (in red) (22) share a similar site on the actin surface. They interact with a patch of hydrophobic amino acids (Tyr-143, Thr-148, Ile-345, Leu-346, Leu-349, Phe-352, and Met-355, yellow) in a cleft between actin subdomains 1 and 3 (green). Gelsolin residues Ala-76, Ala-78, Ile-79, Phe-80, and Val-82 and DBP residues Val-197, Leu-200, and Leu-204, which face the hydrophobic patch on actin, are shown (gray) but not labeled.

Figure 4

All-atom surface representations of the actin–DBP complex. The color scheme is as described for Figs. 1 and 2. Two views of the complex are shown rotated by 90°. The four subdomains of actin are numbered 1–4. All three domains of DBP participate in the interaction with actin subdomains 1 and 3. Notice how the three domains of DBP engulf approximately one half of actin subdomain 3, participating in extensive contacts on both sides of this subdomain.