Function
RIPK1 (Receptor-Interacting Protein Kinase 1) is a protein that plays a central role in cell death and survival pathways, regulating inflammation and maintaining homeostasis. When polyubiquitinated, RIPK1 promotes cell proliferation and differentiation. Here, it acts as part of the TNFR signalling pathway to activate the NF-κB transcription factors. Conversely, in the absence of polyubiquitination, it can form a complex with FADD and caspase 8 to trigger apoptosis. In the case of caspase activity blockage, the assembly of RIPK1/RIPK3 fibrils can trigger necroptosis. The signalling pathway starts with the self-association of RIPK1, leading to the assembly of heteromeric RIPK1-RIPK3 fibrils (canonical necrosome), finally resulting in MLKL oligomerisation to trigger necroptosis.
Disease
RIPK1 has been reported to be involved in many neurodegenerative diseases such as AD, ALS and MS. In Alzheimer's disease, it has been shown to regulate microglial function by modulating cell inflammation [2]. In Amyotrophic Lateral Sclerosis, RIPK1, along with RIPK3 and MLKL, has been shown to contribute to the demyelination and degeneration of neurons [3].
Structural Determination
The primary methods employed to determine the structure of homomeric RIPK1 fibrils were solid-state NMR and electron microscopy. Cryo-probe detection was used in NMR to increases sensitivity by reducing electronic noise. Negative staining electron microscopy was used to capture initial images, while cryo EM was used for further structural determination.
Structural Highlights
RIPK1 has an N-terminal kinase domain, an intervening disordered region and a C-terminal death domain. The disordered region consists of the RHIM (RIP homotypic interaction motif), which is represented by a tetrapeptide with a consensus sequence of (V/I)-Q-(V/I/L/C)-G.
RIPK1 protomers display extended anti-parallel beta-strand architecture, adopting an N-shaped fold within the amyloid core. It consists of three anti-parallel strands, β1 (I529-Y534), β2 (T537-I541), consisting of the RHIM motif and β3 (Y544-I549). β1 and β2 interact via hydrophobic interaction through I531, Y533 and I539 and I541 residues respectively. β2 and β3 establish interaction through a hydrogen bond formed between Q540 and Y546, which is further stabilized by Q540's hydrophobic interactions with M547.
Multiple protomers together form the homomeric RIPK1 fibril. The protomers are found in distinct planes along the fibril axis. The fibril forms a left-handed helical structure having a width of 44 Å, a twist of -7.3° and a pitch of 23.3nm.
The i protomer can interact with the i+1 and i+2 protomers. The I539 residue of β2 of the i protomer establishes hydrophobic contacts with the Y531 residue of the i+1 and i+2 protomers. Similar interactions are present between I541 and T537 of i protomer and I533 and I549 of i+1 and i+2 protomers.
The fibrils also have a ladder of hydrogen bonds that exhibit a stabilizing zipper effect. N545 (β3) of a protomer forms an H-bond with G542 (β2) of the next protomer, locking the β3 of a protomer against the β2 of the next protomer. The N545D mutant showed slower kinetics of fibril formation.
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