A C-RING-like Domain Participates in Protein Self-Assembly and Mineral Nucleation

Abstract

AP7 is a nacre-associated protein of the mollusk shell that forms supramolecular assemblies that nucleate single-crystal aragonite in vitro. AP7 possesses two major sequence regions: a random coil 30-amino acid N-terminal domain (AP7N) and a partially disordered 36-amino acid C-terminal domain (AP7C) that exhibits imperfect sequence homology to the C subclass of the intracellular RING domain family. We report here new findings that implicate the C-RING domain in AP7-mediated supramolecular assembly and single-crystal mineral formation. AP7 protein spontaneously self-assembles over a pH range of 4-9 and is monomeric at pH >9.5. AP7N and AP7C both oligomerize over the pH range of 4-9, with the AP7C sequence closely resembling AP7 in terms of particle morphology and size. In vitro mineralization experiments demonstrate that both AP7N and AP7C form supramolecular assemblies that nucleate single-crystal calcium carbonates. Comparison of previously published nuclear magnetic resonance-based structures of AP7C and AP7N reveals the significant presence of complementary anionic-cationic electrostatic molecular surfaces on AP7C that are not found on AP7N, and this may explain the noted discrepancies between the two domains in terms of self-assembly and single-crystal nucleation. We conclude that the C-RING-like sequence is an important site for AP7 self-association and mineral nucleation, and this represents the first known instance of a RING-like sequence performing these functions within an extracellular protein.

An Oligomeric C-RING Nacre Protein Influences Prenucleation Events and Organizes Mineral Nanoparticles

“…Previously, AP7 self-association was studied as a function of pH using dynamic light scattering (DLS). 22 Understandably, these studies required the use of filtration, and thus, any >0.2 μm protein oligomers, not to mention protein films, would have evaded analyses. Thus, we utilized tapping mode AFM imaging to more definitively monitor apo-AP7 (i.e., calcium-free) oligomerization at pH 8, which corresponds to the pH range for most in vitro calcium carbonate mineralization assays ( Figure 1).…”

“…21,22 Within in vitro settings (carbonate vapor diffusion experiments), AP7 is known to inhibit calcite formation and forms lamellar and clustered single-crystal aragonite. 21,22 However, the efficiency of AP7 in stabilizing aragonite in vitro is low, and the process requires high protein concentrations (i.e., 100 μM). 21 This suggests that AP7 may not function as a true aragonite stabilizer in vivo but perhaps plays other important roles related to nacre tablet formation.…”

“…AP7 also possesses three amyloid-like aggregation-prone cross-β strand domains (Supporting Information, Figure S1), two of which are found within the C-terminal domain. 24 Under mineralization conditions, this interesting combination of disordered and aggregation-prone sequence features leads to the formation of AP7 assemblies that contain mineral deposits 22 and introduce organized nanomineral deposits and subsurface nanoporosities into calcium carbonate crystals. 25 We note that protein oligomerization or aggregation has been documented for other aragonite-associated proteins, 26−29 so the self-associative behavior of AP7 is significant.…”

The multiple effects of amino acids on the early stages of calcium carbonate crystallization

“…This differs with the repetitive domains found in mollusk shell extracellular proteins, which have long repeats of aspartic acid interspersed by a repeat block of valine, alanine, and glutamic acid [24]. It has been thought that these domains found in these proteins are themselves switches which are necessary for structural conformations that are responsible for specific Ca 2þ mineral ion affinities [30]. Interestingly, these domains confer characteristics onto the protein that make it relatively insensitive to temperature induced aggregation, resistance to acid precipitation, and high apparent molecular masses.…”

Ciscem 2014

“…AP7 is an intracrystalline protein found in the nacre layer of the pacific red abalone (Michenfelder et al, 2003). It forms intrinsically instable oligomers that assemble into larger aggregates when calcium ions are present (Amos et al, 2011). Our potentiometric experiments show that this protein influences the progress of the crystallization already in the prenucleation stage by associating with calcium carbonate prenucleation species, thereby prolonging the time till the onset of nucleation (Perovic et al, 2014).…”