The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
PxLPxI/L motif binding to some ANK repeat proteins
Functional site description:
Ankyrin (ANK) repeat domains are one of the more abundant protein modules functioning in protein-protein binding. ANK repeats are comprised of a series of repeating units, with each Ankyrin repeat consisting of 33 amino residues. Ankyrin repeat family A protein 2 (ANKRA2) contains five C-terminal ankyrin repeats and was firstly identified as a binding partner of megalin. The ankyrin repeat domain of ANKRA2 is highly homologous to those of regulatory factor-X associated ankyrin-containing protein (RFXANK). RFXANK plays a critical role in the assembly of a transcriptional complex stimulating MHC II expression. The ankyrin repeat domains of both ANKRA2 and RFXANK interact specifically with PxLPxI/L motifs on diverse binding partners. Thereby, the middle three ankyrin repeats of ANKRA2 and RFXANK form three deep hydrophobic pockets each of which accommodates a single residue of the consensus PxLPxI/L motif.
ELM Description:
The PxLPxI/L motif binds to the ankyrin repeat domains of the two paralogous proteins ANKRA2 and RFXANK. Crystal structures of ANKRA2 and RFXANK in complex with this motif containing proteins revealed binding in an extended binding groove formed by the inner α-helices and inter-repeat hairpin loops of the five ankyrin repeats. Along the groove there are five hydrophobic pockets with the middle three being deepest and most important for the interaction. Each of the three central ankyrin repeats, designated as ANK 2 to 4, harbours a single residue of the PxLPxI/L motif in a hydrophobic pocket so that the binding mode is comparable to a tumbler lock (Xu,2012). In the solved structures there are two permutations of the peptide binding. The first Pro residue of the motif is found in either the first or the second pocket. In all the available structures, at least four of the pockets are occupied by conserved residues of the motif.

The residues between the binding core residues are variable since the respective amino acid side chains point to the solvent, influencing neither binding nor positioning of the conserved binding core. Interestingly, the Ankyrin repeat domain binding partner megalin contains two overlapping PxLPxI/L motifs (Rader,2000). Complex structures (3V20,3V2X) showed an alternative use of both motifs, which supports the consensus role of the core residues for ankyrin repeat binding. Nevertheless, the ankyrin repeat domains of ANKRA2 and RFXANK show specificity for their partners resulting in different binding affinities (Xu,2012, Nie,2015). ANKRA2 has a higher affinity for megalin, CCDC8, RFX7 and the deacetylases, whereas RFXANK is more specific for RFX5. Both surrounding positions and residues within the PxLPxI/L motifs confer binding specificity. Furthermore, a key residue substitution between the ankyrin repeat domains of ANKRA2 and RFXANK, namely the change of H257 of ANKRA2 and R199 of RFXANK, is implicated to dictate specificity upon binding partner selection.
Pattern: P.LP.[IL].{1,3}[VLF]
Pattern Probability: 0.0000267
Present in taxon: Eumetazoa
Interaction Domains:
o See 10 Instances for LIG_ANK_PxLPxL_1
o Abstract
Ankyrin repeat domains (PF00023) are a common protein module in eukaryotic proteomes. They are named after the archetypal protein ankyrin, a key component of the membrane-associated cystoskeleton.They are comprised of short, tandem repeating motifs, with variable repeat numbers ranging from 2 to 34. Each basic repeating core unit consists of 33 amino residues and adopts a canonical helix-turn-helix conformation (15152081). The repeats are arranged head to tail, forming into rods of variable lengths.

ANKRA2 (3SO8) contains five ankyrin repeats at its C-terminus and was initially identified as interaction partner of megalin, which is also known as low-density lipoprotein receptor-related protein 2 (LRP2) (Rader,2000). Megalin is a multiligand-binding receptor located in the plasma membrane of absorptive epithelial cells (11994745). Via yeast two-hybrid screening, ANKRA2 was further characterized as a binding partner of the class IIa histone deacytelases (HDACs) 4 and 5 (Wang,2005, McKinsey,2005). Class IIa HDACs and the cytoplasmic tail of megalin share a conserved PxLPxI/L motif, which mediates specific binding to the ankyrin repeat domain of ANKRA2 (3V20, 3V2X, 3V31). The motif is also found in the alternatively spliced isoform 2 of ABL1. ANKRA2 became also implicated in the 3M syndrome, a primordial growth disorder, since its ankyrin repeat domain interacts with the PxLPxI/L motif located in the C-terminal region of the coiled-coil domain containing 8 (CCDC8) protein (Nie,2015, 4LG6). Nevertheless, the exact biological relevance of ANKRA2 remains elusive.

The ankyrin repeats of ANKRA2 are homologous to those of RFXANK (Krawczyk,2005), sharing 62% sequence identity. RFXANK functions in the positive regulation of major histocompatibility class II (MHC II) gene expression since it associates with RFX5 and RFXAP to form a trimeric transcription factor complex essential for the recruitment of transcriptional co-activators. Mutations in the RFXANK gene cause the immune disorder bare lymphocyte syndrome (BLS). RFXANK is also shown to bind to the transcriptional repressors HDAC4 and HDAC5 (Wang,2005, McKinsey,2005). The functional consequences of RFXANK-HDAC interaction are not well-characterized.

Similar to the ANKRA2 interaction with histone deacetylases and megalin, the ankyrin repeat domain of RFXANK recognizes its binding partners via a consensus PxLPxI/L motif. Therefore, the ankyrin repeat domains of both ANKRA2 and RFXANK bind specifically to PxLPxI/L motifs found in cellular proteins (Xu,2012).

Crystal structure analysis showed that the ankyrin repeat domains of ANKRA2 and RFXANK have a groove with five hydrophobic pockets. The deepest pockets are in the central repeats and they accommodate the core motif residues of the PxLPxI/L motif. A fourth pocket is also occupied by a less conserved hydrophobic residue. The use of hydrophobic pockets along an extended binding groove is likened to a tumbler-lock binding mode (Xu,2012). ANKRA2 and RFXANK bind to PxLPxI/L motif containing proteins with different binding affinities and hence may display specificity for their partners.
o 6 selected references:

o 10 GO-Terms:

o 10 Instances for LIG_ANK_PxLPxL_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P40238 MPL
530 539 RHALWPSLPDLHRVLGQYLR TP 1 Homo sapiens (Human)
P00519-2 ABL1
15 22 LGDQRRPSLPALHFIKGAGK TP 1 Homo sapiens (Human)
500 509 AFWHTPRLPTLPKRVPRAGE TP 5 Homo sapiens (Human)
91 98 KAFVHMPTLPNLDFHKTGDG TP 3 Homo sapiens (Human)
P48382 RFX5
173 180 KTLVSMPPLPGLDLKGSESP TP 5 Homo sapiens (Human)
P98158 Lrp2
4460 4468 LLPTLPKLPSLSSLAKPSEN TP 10 Rattus norvegicus (Norway rat)
325 334 SLYTSPSLPNITLGLPAVPS TP 1 Homo sapiens (Human)
Q9Z2V6 Hdac5
358 367 SLYTSPSLPNISLGLQATVT TP 2 Mus musculus (House mouse)
367 376 SLYTSPSLPNISLGLQATVT TP 6 Homo sapiens (Human)
P56524 HDAC4
349 358 PLYTSPSLPNITLGLPATGP TP 14 Homo sapiens (Human)
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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