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Elasticity and rupture of a multi-domain neural cell adhesion molecule complex - PubMed

  • ️Thu Jan 01 2009

Elasticity and rupture of a multi-domain neural cell adhesion molecule complex

Venkat Maruthamuthu et al. Biophys J. 2009.

Abstract

The neural cell adhesion molecule (NCAM) plays an important role in nervous system development. NCAM forms a complex between its terminal domains Ig1 and Ig2. When NCAM of cell A and of cell B connect to each other through complexes Ig12(A)/Ig12(B), the relative mobility of cells A and B and membrane tension exerts a force on the Ig12(A)/Ig12(B) complex. In this study, we investigated the response of the complex to force, using steered molecular dynamics. Starting from the structure of the complex from the Ig1-Ig2-Ig3 fragment, we first demonstrated that the complex, which differs in dimensions from a previous structure from the Ig1-Ig2 fragment in the crystal environment, assumes the same extension when equilibrated in solvent. We then showed that, when the Ig12(A)/Ig12(B) complex is pulled apart with forces 30-70 pN, it exhibits elastic behavior (with a spring constant of approximately 0.03 N/m) because of the relative reorientation of domains Ig1 and Ig2. At higher forces, the complex ruptures; i.e., Ig12(A) and Ig12(B) separate. The interfacial interactions between Ig12(A) and Ig12(B), monitored throughout elastic extension and rupture, identify E16, F19, K98, and L175 as key side chains stabilizing the complex.

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Figures

Figure 1
Figure 1

(A) Schematic of the extracellular part of NCAM showing the arrangement of its different domains. (B) Structure of the complex formed between Ig12 of molecule A (dark gray) and Ig12 of molecule B (light gray) in cartoon representation (bottom right). The view on the left shows the two main aromatic residues of Ig1(A) that bind to their pockets in Ig2(B) (surface representation). The view on the top right shows the interdomain, intramolecule bonds in the linker region between E16 (of Ig1) and K98 (of Ig2).

Figure 2
Figure 2

Time evolution of the RMSD of the complex during the minimization and equilibration steps before releasing the restraints on the SMD atom.

Figure 3
Figure 3

Variation of the end-to-end length and the RMSD of the complex (relative to that at time t = 0) with time, after releasing the restraints on the SMD atom. Inset: The conformations of the complex before (black) and after (gray) this final equilibration step, shown superimposed.

Figure 4
Figure 4

(A) Initial state of the intramolecule interdomain bonds in molecule A (similar in molecule B) before equilibration. (B) Hydrogen bonding partners of the aromatic residues Y65 (K133, E171, and R173) and F19 (G178) in the Ig1(A)/Ig2(B) interface.

Figure 5
Figure 5

(A) Evolution of the end-to-end length of the complex with time, when subjected to constant forces of 50 and 70 pN. (B) The resting conformation (surface representation), shown in gray, and the conformation of the complex when subjected to 50 pN force shown in black (cartoon representation). (C) Plot showing the dependence of the increase in end-to-end length of the complex (relative to the unstrained complex) on the applied force.

Figure 6
Figure 6

Identification of a loading rate for unbinding: response of the Ig12/Ig12 complex to pulling at two computationally realizable loading rates. At the two loading rates, the complex initially stretches to almost twice its end-to-end length, after which its response depends on the loading rate. The complex unfolds as it unbinds at a loading rate of 7 pN/ps (v = 0.1 Å/ps), but unbinds without unfolding at 0.7 pN/ps (v = 0.01 Å/ps).

Figure 7
Figure 7

Unbinding trajectory of the complex during 0.01 Å/ps constant velocity pulling. All images show the binding interface between Ig1(A) and Ig2(B). (A) By 1.75 ns, Y65's H-bond with K133 is already broken, whereas its H-bonds with E171 and R173 are about to break. (B) Even at 4 ns, F19 remains bound to its complementary pocket in Ig2, as shown. The H-bond between the backbone −NH of F19 and G178 is also shown. (C) By 4.45 ns, E16 and K98, which are interdomain intramolecule H-bonds initially, have ruptured and formed intermolecule bonds instead. (D) By 5 ns, F19 is only loosely bound to its now half-open pocket in Ig2. (E) As F19 slips away from its initial binding pocket, L175 binds to a nonpolar pocket (white-colored region) of Ig1 (colored according to residue type in surface representation) formed by V6, P7, F19, and L21 as pictured at 5.18 ns. Its rupture 0.1 ns later forms the final rupture event (marked “3” in F). (F) Time evolution of the force experienced by the complex and the bond distance of the Y65(A)-R173(B), I12(A)-F96(A), and F19(A)-G178(B) H-bonds. The major force peaks are marked 1, 2, and 3. Ig1A is shown in blue and Ig2B in red in A–D. Residue structures in A–E are shown in licorice representation with the atom-based color-codes N-blue, C-cyan, O-red, and H-white.

Figure 8
Figure 8

Schematic illustration of the nanomechanical spring-like behavior of NCAM when bound as an Ig12/Ig12 trans complex. Note that Ig domains 3–5, the putative hinge, and the two Fn III domains are lumped together in this schematic and that they also contribute to the effective spring constant.

Figure 9
Figure 9

Complementary H-bond formation between E16(A) and K98(B) (inset). The distance between each of the 3 hydrogens of lysine's −NH3+ and the 2 oxygens of glutamate's −COO- show that when any one H-bond breaks, another one forms and that they do so in a mutually exclusive, but complementary, way between 1.2 and 4.85 ns.

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