Modular Organization and Combinatorial Energetics of Proline–Tyrosine Nuclear Localization Signals

Süel, Katherine E.; Gu, Hongmei; Chook, Yuh Min

doi:10.1371/journal.pbio.0060137

Cited by 75 publications

(160 citation statements)

References 77 publications

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“…Extensive mutagenesis and thermodynamic analyses corroborated structural findings that the consensus motifs form three linker-separated binding epitopes (Fig. 1A) (21). Epitope 1 is the hydrophobic or basic N-terminal motif; epitopes 2 and 3 are the arginine residue and the PY dipeptide of the C-terminal RX 2-5 PY motif, respectively.…”

supporting

confidence: 67%

“…Epitope 1 is the hydrophobic or basic N-terminal motif; epitopes 2 and 3 are the arginine residue and the PY dipeptide of the C-terminal RX 2-5 PY motif, respectively. Each epitope can contribute very differently to total binding energy in different PY-NLSs and signal diversity can be achieved through combinatorial mixing of energetically weak and strong motifs while maintaining affinity appropriate for nuclear import (21).…”

mentioning

confidence: 99%

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

Zhang

Chook²

2012

Proc. Natl. Acad. Sci. U.S.A.

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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. ...

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supporting

confidence: 67%

mentioning

confidence: 99%

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

Zhang

Chook²

2012

Proc. Natl. Acad. Sci. U.S.A.

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170

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“…35 VKKPHR 40 (possible bipartite c-NLS consensus residues are underlined), does not match the bipartite c-NLS consensus of (K/R)(K/R)X 10 -12 (K/ R)3/5) because linkers between basic segments are either too short or too long. If the bipartite NLS is 17 40 , the 16-residue linker is too long. Imp␣ can recognize bipartite c-NLSs with artificial linkers as short as eight residues and linkers up to 40 residues (32, 48 -51).…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, the energetic contribution of the IK-NLS motif to Imp5 binding is less than that of all basic residues within residues 11-27 as the MBP-H3 tail(K14A/K17A/K18A/K23A/R26A/K27A) mutant decreases Imp5 affinity by 15-fold (Table 1). Mutation of both the basic segment spanning residues 11-27 and the IK-NLS epitope (MBP-H3 tail(K14/K17/K18/K23/R26/K27/ 35 VK-KPHR 40 to Ala) mutant) completely abolishes Imp5 binding (Table 1 and Fig. 3B).…”

Section: Seven Different Human Importins Bind the H3 And H4mentioning

confidence: 95%

Recognition Elements in the Histone H3 and H4 Tails for Seven Different Importins

Soniat

Çağatay

Chook

2016

Journal of Biological Chemistry

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N-terminal tails of histones H3 and H4 are known to bind several different Importins to import the histones into the cell nucleus. However, it is not known what binding elements in the histone tails are recognized by the individual Importins. Biochemical studies of H3 and H4 tails binding to seven Importins, Imp␤, Kap␤2, Imp4, Imp5, Imp7, Imp9, and Imp␣, show the H3 tail binding more tightly than the H4 tail. The H3 tail binds Kap␤2 and Imp5 with K D values of 77 and 57 nM, respectively, and binds the other five Importins more weakly. Mutagenic analysis shows H3 tail residues 11-27 to be the sole binding segment for Imp␤, Kap␤2, and Imp4. However, Imp5, Imp7, Imp9, and Imp␣ bind two separate elements in the H3 tail: the segment at residues 11-27 and an isoleucine-lysine nuclear localization signal (IK-NLS) motif at residues 35-40. The H4 tail also uses either one or two basic segments to bind the same set of Importins with a similar trend of relative affinities as the H3 tail, albeit at least 10-fold weaker. Of the many lysine residues in the H3 and H4 tails, only acetylation of the H3 Lys 14 substantially decreased binding to several Importins. Lastly, we show that, in addition to the N-terminal tails, the histone fold domains of H3 and H4 and/or the histone chaperone Asf1b are important for Importin-histone recognition.New nucleosomes are assembled in the nucleus during S phase as new core histones H2A, H2B, H3, and H4 are synthesized, assembled into H2A/H2B and H3/H4 dimers in the cytoplasm, and then imported into the nucleus for deposition onto replicating chromatin (1-8). Little is known about cytoplasmic assembly/processing of H2A and H2B, but assembly/processing of H3 and H4 are better understood. In the cytoplasm, H3 and H4 are passed from one histone chaperone to another for folding, assembly into H3/H4 dimers, and acetylation.

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“…Co-crystal structures of Kapb2 in complex with an NLS peptide from its bestcharacterized cargo protein, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), reveal that the negatively-charged NLS binding domain of Kapb2 interacts with a long ([30 amino acids) structurally disordered basic sequence containing a proline (P) and tyrosine (Y) in hnRNP A1 (Table 1). Chook et al defined the PY-NLS as a consensus sequence containing a hydrophobic or basic region followed by an arginine (R)/lysine (K)/histidine (H) then a proline and tyrosine (R/K/H-X (2-5) -PY) (35,62). Using these guidelines, the human proteome was searched and 81 potential cargoes for Kapb2 were identified, (49,50) some of which they demonstrated interact directly with Kapb2 in vitro (35).…”

Section: Proline-tyrosine Nls (Py-nls) Sequencesmentioning

confidence: 99%

Nuclear localization signals and human disease

McLane

Corbett

2009

IUBMB Life

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SummaryIn eukaryotic cells, the physical separation of the genetic material in the nucleus from the translation and signaling machinery in the cytoplasm by the nuclear envelope creates a requirement for a mechanism through which macromolecules can enter or exit the nucleus as necessary. Nucleocytoplasmic transport involves the specific recognition of cargo molecules by transport receptors in one compartment followed by the physical relocation of that cargo into the other compartment through regulated pores that perforate the nuclear envelope. The recognition of protein cargoes by their transport receptors occurs via amino acid sequences in cargo proteins called nuclear targeting signals. Both nuclear import and export of proteins are highly regulated processes that control, not only what cargo can enter and/or exit the nucleus, but also when in the cell cycle and in what cell type, the cargo can be transported. Deregulation of the nuclear transport of specific cargoes has been linked to numerous cancers and developmental disorders highlighting the importance of understanding the mechanisms underlying nucleocytoplasmic transport and particularly the modulation of the specific interactions between transporter receptors and nuclear targeting signals within target cargo proteins.2009 IUBMB IUBMB Life, 61(7): [697][698][699][700][701][702][703][704][705][706] 2009

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Modular Organization and Combinatorial Energetics of Proline–Tyrosine Nuclear Localization Signals

Cited by 75 publications

References 77 publications

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

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

Recognition Elements in the Histone H3 and H4 Tails for Seven Different Importins

Nuclear localization signals and human disease

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