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

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.

The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity

“…The pNLS sequence plausibly also modulates binding of NLS to importin ␣ and hence the kinetics of nuclear import. This argument is supported by recent work by Smith et al (46) who showed that a hydrophobic residue at the "P4" position of a monopartite NLS (such as methionine at the KKMK motif) is generally unfavored, and one of the strategies to remediate such a detrimental effect is the presence of positive charges upstream of the NLS.…”

Application of In Silico and HTS Approaches to Identify Nuclear Import Inhibitors for Venezuelan Equine Encephalitis Virus Capsid Protein: A Case Study

“…The crystal structure was based on a 12amino-acid peptide containing the VEEV CP nuclear localization signal (NLS) recognized by Impα. However, the classical Impα binding NLS is only a short K-K/R-X-K/R sequence (Marfori et al, 2012;Smith et al, 2018). Docking simulations of truncated versions of the VEEV CP peptide to Impα identified the minimal region required to model Impα binding as the core of the NLS (KKPK; amino acids 6-9) using estimates of the free energy of binding.…”

N-myristoyltransferase: Tracing Steps Backwards to Find a Way Forward

“…Whereas a non-classical NLS comprises of proline-tyrosine (PY) stretch of residues. Classical NLS can be either monopartite comprising a single stretch of basic amino acids as reported in SV40 large T-antigen [41] or bipartite comprising two basic stretches of amino acids separated by a spacer sequence as observed in Ty1 integrase [42]. The non-classical NLS includes the PY sequence containing motif as reported in the Hrp1 protein found in Saccharomyces cerevisiae [43] and the M9 sequence containing motif as reported in the splicing factor, HnRNP A1.…”