Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting

Kate M. Smith, Veronica Di Antonio, Luca Bellucci, David R. Thomas, Fabiana Caporuscio, Francesco Ciccarese, Hanieh Ghassabian, Kylie M. Wagstaff, Jade K. Forwood, David A. Jans, Giorgio Palù, Gualtiero Alvisi

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

Original languageEnglish
Pages (from-to)1114-1129
Number of pages16
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1865
Issue number8
DOIs
Publication statusPublished - 1 Aug 2018

Keywords

  • Importin alpha
  • IMPs
  • NLS
  • Nuclear import
  • Nuclear transport
  • T-ag

Cite this

Smith, Kate M. ; Di Antonio, Veronica ; Bellucci, Luca ; Thomas, David R. ; Caporuscio, Fabiana ; Ciccarese, Francesco ; Ghassabian, Hanieh ; Wagstaff, Kylie M. ; Forwood, Jade K. ; Jans, David A. ; Palù, Giorgio ; Alvisi, Gualtiero. / Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting. In: Biochimica et Biophysica Acta - Molecular Cell Research. 2018 ; Vol. 1865, No. 8. pp. 1114-1129.
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abstract = "Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.",
keywords = "Importin alpha, IMPs, NLS, Nuclear import, Nuclear transport, T-ag",
author = "Smith, {Kate M.} and {Di Antonio}, Veronica and Luca Bellucci and Thomas, {David R.} and Fabiana Caporuscio and Francesco Ciccarese and Hanieh Ghassabian and Wagstaff, {Kylie M.} and Forwood, {Jade K.} and Jans, {David A.} and Giorgio Pal{\`u} and Gualtiero Alvisi",
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Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting. / Smith, Kate M.; Di Antonio, Veronica; Bellucci, Luca; Thomas, David R.; Caporuscio, Fabiana; Ciccarese, Francesco; Ghassabian, Hanieh; Wagstaff, Kylie M.; Forwood, Jade K.; Jans, David A.; Palù, Giorgio; Alvisi, Gualtiero.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1865, No. 8, 01.08.2018, p. 1114-1129.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Contribution of the residue at position 4 within classical nuclear localization signals to modulating interaction with importins and nuclear targeting

AU - Smith, Kate M.

AU - Di Antonio, Veronica

AU - Bellucci, Luca

AU - Thomas, David R.

AU - Caporuscio, Fabiana

AU - Ciccarese, Francesco

AU - Ghassabian, Hanieh

AU - Wagstaff, Kylie M.

AU - Forwood, Jade K.

AU - Jans, David A.

AU - Palù, Giorgio

AU - Alvisi, Gualtiero

PY - 2018/8/1

Y1 - 2018/8/1

N2 - Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

AB - Nuclear import involves the recognition by importin (IMP) superfamily members of nuclear localization signals (NLSs) within protein cargoes destined for the nucleus, the best understood being recognition of classical NLSs (cNLSs) by the IMPα/β1 heterodimer. Although the cNLS consensus [K-(K/R)-X-(K/R) for positions P2–P5] is generally accepted, recent studies indicated that the contribution made by different residues at the P4 position can vary. Here, we apply a combination of microscopy, molecular dynamics, crystallography, in vitro binding, and bioinformatics approaches to show that the nature of residues at P4 indeed modulates cNLS function in the context of a prototypical Simian Virus 40 large tumor antigen-derived cNLS (KKRK, P2–5). Indeed, all hydrophobic substitutions in place of R impaired binding to IMPα and nuclear targeting, with the largest effect exerted by a G residue at P4. Substitution of R with neutral hydrophobic residues caused the loss of electrostatic and van der Waals interactions between the P4 residue side chains and IMPα. Detailed bioinformatics analysis confirmed the importance of the P4 residue for cNLS function across the human proteome, with specific residues such as G being associated with low activity. Furthermore, we validate our findings for two additional cNLSs from human cytomegalovirus (HCMV) DNA polymerase catalytic subunit UL54 and processivity factor UL44, where a G residue at P4 results in a 2–3-fold decrease in NLS activity. Our results thus showed that the P4 residue makes a hitherto poorly appreciated contribution to nuclear import efficiency, which is essential to determining the precise nuclear levels of cargoes.

KW - Importin alpha

KW - IMPs

KW - NLS

KW - Nuclear import

KW - Nuclear transport

KW - T-ag

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U2 - 10.1016/j.bbamcr.2018.05.006

DO - 10.1016/j.bbamcr.2018.05.006

M3 - Article

VL - 1865

SP - 1114

EP - 1129

JO - Biochimica et Biophysica Acta - Molecular Cell Research

JF - Biochimica et Biophysica Acta - Molecular Cell Research

SN - 0167-4889

IS - 8

ER -