Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

Abstract

Mutations in the proline/tyrosine-nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif, a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (K D for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUSKapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.T he proline/tyrosine-nuclear localization signal (PY-NLS) of RNA-binding protein Fused in Sarcoma (FUS) was first identified in a structure-based bioinformatics approach and shown to bind the import-karyopherin, karyopherinβ2 (Kapβ2) in a Ran-sensitive manner (1). More recently, the signal was shown to direct Kapβ2-mediated nuclear import in cells (2) and to be heavily mutated in ∼5% of familial amyotrophic lateral sclerosis (ALS) (3-5), a progressive and fatal neurodegenerative disorder. ALS mutations in the PY-NLS disrupted nuclear import of FUS, causing its mislocalization and aggregation in the cytoplasm, as evidenced by cytoplasmic FUS inclusions in motor neurons of ALS patients (2, 6-9). The PY-NLS of FUS is also mutated in another neurodegenerative disease, frontotemporal lobar dementia (FTLD), which also is characterized by cytoplasmic mislocalization and aggregation of FUS (10, 11). The pathogenic role of FUS PY-NLS mutants was further confirmed by observations that expression of such proteins in Drosophila, Caenorhabditis elegans, zebrafish, and rats caused neurodegeneration (12-15). Proper FUS localization is important in maintaining neuronal homeostasis, and it is thus important to understand the factors that govern localization of both wild-type and mutant proteins.PY-NLSs are recognized by the import-karyopherin Kapβ2 (also known as Transportin) for transport through the nuclear pore complex into the nucleus (1, 16-18). These 15-to 100-residue-long sequences are large and diverse and cannot be sufficiently described by a traditional consensus sequence but are instead described by a collection of physical rules that include requirements for intrinsic structural disorder, overall basic character, and a set of sequence motifs. PY-NLS motifs consist of an N-terminal hydrophobic or basic motif and a C-terminal RX 2-5 PY motif (Fig. ...

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

“…In vitro methylation of recombinant PABPN1 by PRMT1 was sufficient to interfere with binding to recombinant transportin (Fronz et al , 2011 ). This is in agreement with crystal structures displaying contacts between transportin and arginine side chains in PY-NLSs from other cargo proteins (Lee et al , 2006 ;Cansizoglu et al , 2007 ;Imasaki et al , 2007 ;Zhang and Chook , 2012 ). Competition assays with the methylated and unmethylated NLS peptide showed that methylation reduces the affinity for transportin about 10-fold.…”

“…The 61-amino-acid-long C-terminal domain of nucleolin, a prototype RGG domain, lacks stable secondary or tertiary structure, at least in isolation (Zahariev et al , 2005 ). The C-terminal domain of PABPN1 can safely be assumed to lack stable structure, as it is a binding site for transportin (Calado et al , 2000a ;Fronz et al , 2011 ), which is known to bind its cargo in an unfolded or mostly unfolded conformation (Lee et al , 2006 ;Cansizoglu et al , 2007 ;Imasaki et al , 2007 ;Zhang and Chook , 2012 ).…”

A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1

“…There is no PY in the ADAR1 C-terminal module, but our structural model shows that residues 800 ER 801 could occupy the corresponding Trn1 pocket and therefore represent a rather degenerated equivalent to epitope 3. Interestingly, although the PY energetic contribution is sometimes important and explains the sensitivity to mutations in this motif (e.g., hnRNP M, hnRNP D, and FUS/TLS) (9,10,38), the contribution of epitope 3 to the overall binding affinity is sometimes relatively small and epitope 3 is then tolerant to mutations (11). In the case of ADAR1-NLS, however, Arg801 is essential for Trn1 binding and is thus an important constituent for nuclear import.…”

“…5 A and B). Among all NLS peptides, we drew a particular attention to the fused in sarcoma/translocated in sarcoma (FUS/TLS) NLS because its central region adopts an α-helical structure forming approximately two helical turns (Gly515-Arg521; PDB ID code 4FDD) (38). The two helical turns could create a spacer that maintains the N-and C-terminal modules at the same distance as in the original dsRBD context (SI Appendix, Fig.…”

Activity-dependent FUS dysregulation disrupts synaptic homeostasis

“…Although FUS R521G is not overtly mislocalized in our animals, it is likely that the shuttling dynamics of FUS R521G are altered, because FUS R521 residue is located at PY-NLS. On the other hand, PY-NLS has very high binding affinity (k d = 9.5 nM) for karyopherinβ2 (Kapβ2, also known as "transportin"), which mediates FUS nuclear shuttling (37). Although ALS mutations in PY-NLS reduce Kapβ2-binding affinities by several fold (37), the mutant FUS proteins still have nanomolar affinity and thus are expected to be translocated efficiently to the nucleus unless the nuclear import machinery is overwhelmed (such as when wild-type or mutant FUS proteins are massively overexpressed).…”