Volume 23 September 15, 2012
MBoC | ARTICLE
NESdb: a database of NES-containing CRM1
Darui Xua, Nick V. Grishinb, and Yuh Min Chooka
aDepartment of Pharmacology and bHoward Hughes Medical Institute and Department of Biochemistry, University of
Texas Southwestern Medical Center at Dallas, Dallas, TX 75390
ABSTRACT The leucine-rich nuclear export signal (NES) is the only known class of targeting
signal that directs macromolecules out of the cell nucleus. NESs are short stretches of
8–15 amino acids with regularly spaced hydrophobic residues that bind the export kary-
opherin CRM1. NES-containing proteins are involved in numerous cellular and disease pro-
cesses. We compiled a database named NESdb that contains 221 NES-containing CRM1
cargoes that were manually curated from the published literature. Each NESdb entry is an-
notated with information about sequence and structure of both the NES and the cargo
protein, as well as information about experimental evidence of NES-mapping and CRM1-
mediated nuclear export. NESdb will be updated regularly and will serve as an important
resource for nuclear export signals. NESdb is freely available to nonprofit organizations at
Dynamic nuclear–cytoplasmic trafficking of macromolecules con-
trols many eukaryotic cellular processes, such as gene expression,
signal transduction, cell differentiation, and immune response. The
karyopherin-β family of transport factors recognizes targeting sig-
nals within cargo proteins for transport in and out of the nucleus.
Nuclear localization signals direct proteins into the nucleus, and
nuclear export signals (NESs) direct proteins into the cytoplasm (re-
viewed in Görlich and Kutay, 1999; Chook and Blobel, 2001; Conti
and Izaurralde, 2001; Weis, 2003; Kutay and Güttinger, 2005; Tran
et al., 2007; Xu et al., 2010).
The leucine-rich or classic NES is the only class of nuclear export
signal that has been characterized. An NES is 8–15 amino acids
long and contains regularly spaced hydrophobic residues. The
name leucine-rich NES was coined because the first signals identi-
fied in the HIV-1 Rev and PKIα proteins are enriched with leucine
residues (Fischer et al., 1994; Meyer and Malim, 1994; Wen et al.,
1995). Since then, many more NES-containing proteins have been
identified, and mutagenesis and computational analyses have
shown the NES sequences to be more diverse and conform to the
loose consensus sequence φ-X2-3-φ-X2-3-φ-X-φ, where φ is L, V, I, F,
or M and X is any amino acid (Bogerd et al., 1996; Henderson and
Eleftheriou, 2000; Engelsma et al., 2004; la Cour et al., 2004; Kutay
and Güttinger, 2005). The NES is recognized by the export kary-
opherin CRM1, which is also known as exportin 1 (Fornerod et al.,
1997; Fukuda et al., 1997; Neville et al., 1997; Ossareh-Nazari
et al., 1997; Richards et al., 1997; Stade et al., 1997). Recently pub-
lished crystal structures of CRM1 bound to several NESs showed
that the signals adopt either combined α-helix–loop or all-loop
structures that bind in a hydrophobic groove on the convex surface
of CRM1 (Dong et al., 2009a,b; Monecke et al., 2009; Güttler et al.,
2010). Leptomycin B (LMB) inhibits nuclear export by forming a
covalent bond with Cys528 of human CRM1, which is located in the
NES-binding groove, thus blocking access of the NES to its binding
site (Kudo et al., 1999; Dong et al., 2009b; Monecke et al., 2009).
NESs have been identified in >300 proteins with diverse func-
tions, such as transcription factors, cell cycle regulators, ribonucleo-
protein complexes, translation factors, and viral proteins (Fischer
et al., 1994; Wen et al., 1995; Fridell et al., 1996; Ho et al., 2000;
Murdoch et al., 2002; Vissinga et al., 2009). Nuclear export of viral
proteins by CRM1 is important for replication of many viruses that
cause human diseases. Aberrant mislocalization of cellular CRM1
cargoes also interrupts numerous cellular processes, often resulting
in diseases. Therefore controlling CRM1–NES interactions might be
a potential therapeutic target for many disease conditions such as
Salk Institute for Biological
Received: Jan 23, 2012
Revised: May 16, 2012
Accepted: Jul 16, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E12-01-0045) on July 25, 2012.
Address correspondence to: Yuh Min Chook (yuhmin.chook@utsouthwestern
Abbreviations used: CRM1, chromosome region maintenance 1; HIV, human
immunodeficiency virus; LMB, leptomycin B; NES, nuclear export signal; PKIα,
cAMP-dependent protein kinase inhibitor alpha.
© 2012 Xu et al. This article is distributed by The American Society for Cell Biol-
ogy under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
3674 | D. Xu et al. Molecular Biology of the Cell
a search button, which searches the full name, alternative names,
and organism of proteins for the keywords. Clicking on a particular
protein will load the individual page for the protein.
Each entry contains 14 features related to the sequence, struc-
ture, and nuclear export activity of the NESs and cargo proteins. A
sample page for snurportin 1 (SNUPN) is shown in Figure 1. The
NES features include the following:
Full name: the recommended name in UniProt database for the
given protein, along with the link to its entry in UniProt (Jain
et al., 2009; The UniProt Consortium, 2011).
Alternative names: other names of the protein that are com-
monly used in the literature.
Organism: the organism of the listed protein.
Experimental evidence for CRM1-mediated nuclear export:
reported experimental evidence for CRM1-mediated nuclear
export. Reports on whether 1) the protein binds CRM1, 2) the
protein is retained in the nucleus by LMB, 3) nuclear export of the
protein is affected by another CRM1 cargo such as the HIV-Rev
protein, or 4) CRM1 is required for nuclear export in digitonin-
permeabilization transport assays. All NESdb entries contain
experimental evidence for CRM1-mediated nuclear export.
Mutations that affect nuclear export: mutations that have been
shown to disrupt nuclear export in cells.
Mutations that affect CRM1 binding: mutations that have been
shown to disrupt in vitro CRM1 binding.
Functional export signals: the protein segment that resembles an
NES and when fused to a reporter protein can independently
target the reporter for nuclear export. Such data define boundar-
ies and sufficiency of the putative minimal NESs.
Secondary structure of the export signal: the explicitly denoted,
experimentally determined secondary structure for the reported
Other residues important for export: residues known to contact
CRM1 or shown to affect CRM1 binding or nuclear export but are
located outside of the NES segment. This information may be
useful since cargoes may bind CRM1 in multipartite manner and
contain additional binding epitopes.
Sequence: sequence of the full-length protein with the functional
NES underlined. Mutations that disrupt nuclear export in cells are
highlighted in yellow, mutations that disrupt in vitro CRM1 bind-
ing are in red, and other, non-NES residues reported to affect
CRM1 binding or nuclear export are in green. There are four tabs
associated with the sequence: 1) the sequence in FASTA format
(Pearson and Lipman, 1988), 2) conserved domains of the protein
obtained from the Conserved Domain Database (Marchler-Bauer
et al., 2009), 3) predicted secondary structure of the protein by
PSIPRED (McGuffin et al., 2000), and 4) conservation scores of
the protein calculated by AL2CO (Pei and Grishin, 2001).
Three-dimensional structures: links to three-dimensional struc-
tures in the Protein Data Bank (PDB), if available.
Comments: a short summary of protein functions and experi-
ments related to the identification of its transport signals.
References: the literature from which the features were extracted,
with links to PubMed.
User input: because NESs are easily misidentified, a user input
field at the bottom of each entry that allows users to positively or
negatively flag the NES after submitting supporting comments/
cancer and viral infections (Bogerd et al., 1995; Yi et al., 2002;
Faustino et al., 2007; Noske et al., 2008).
A database of 80 NESs named NESbase 1.0 was compiled in
2003 (la Cour et al., 2003). More recently, Fu et al. (2011) published
a list of 70 NES-containing proteins. Here, we present NESdb, an
up-to-date and substantially larger NES database with 221 experi-
mentally identified entries. Each entry is annotated with many de-
tailed features related to the sequence, structure, and nuclear export
activity of the NESs and cargo proteins. NESdb is a valuable informa-
tion resource for the biomedical research community to learn about
nuclear export signals that have already been identified. Analysis of
the sequences and three-dimensional structures of NESs in NESdb
and false-positive NESs generated from NESdb revealed some dis-
tinguishing features that might be important for the future develop-
ment of accurate NES prediction algorithms (Xu et al., 2012).
DATABASE CONTENT AND DEVELOPMENT
NESdb contains 221 entries as of December 2011. Each entry is a
protein that contains one or more NESs. All NESs listed in NESdb
were experimentally identified and reported in the published litera-
ture. Both the PubMed and UniProt databases were searched using
keywords “nuclear export signal,” “NES,” and “CRM1” (Jain et al.,
2009; The UniProt Consortium, 2011). The returned literature was
examined with the following criteria to identify the existence of an
experimentally tested NES: 1) evidence of CRM1-dependent nu-
clear export, such as binding to CRM1, inhibition by LMB, nuclear
retention at nonpermissive temperature in CRM1 temperature-
sensitive yeast strains, or competition with other CRM1 cargoes;
2) the presence of a protein segment that matches the traditional
NES consensus sequence φ-X2-3-φ-X2-3-φ-X-φ, which can target a re-
porter protein for nuclear export; and 3) the presence of mutations
within the tested NES segment that abolished nuclear export of the
full-length protein. All proteins in NESdb meet the first criterion, and
many meet all three criteria. The collected information is manually
entered into the database. NESdb was implemented as a MySQL
database. PHP5 was used to connect to the database and dynami-
cally generate HTML pages. Apache Web server hosted on a Linux
cluster was used to serve the database.
DATABASE ACCESS AND USER INTERFACE
The NESdb database is freely available for nonprofit organizations
at http://prodata.swmed.edu/LRNes. At this time, NESdb contains
221 experimentally identified CRM1 cargoes reported in the litera-
ture. The published literature is searched on a bimonthly basis and
NESdb is updated with every 20 new entries. However, many se-
quences in the genome, especially those in amphipathic helices,
match the NES consensus, thus making accurate NES identification
difficult. It is likely that some published studies contain mistakenly
identified NESs. As a caution to the research community, we sepa-
rated the 221 proteins in NESdb into two groups. The first group is
named “NESs” and contains experimentally identified NESs with no
contradicting experimental evidence. The second group is named
“NESs in doubt” and contains proteins that were initially reported as
NESs but with doubts on their validity cast by subsequent experi-
ments. Clicking the corresponding link on the main page brings up
a list of proteins that belongs to each group. The list can be sorted
alphabetically by protein names or numerically by protein ID num-
bers in NESdb. Users are able to positively or negatively flag specific
NES-containing proteins on their individual pages. A tally of flags for
each protein is displayed next to its name on the list. An entry with
many negative flags will be reevaluated and moved to the “NESs in
doubt” category or vice versa. The database is also equipped with
Volume 23 September 15, 2012 NESdb, an NES database | 3675
NESdb will contribute to the understanding of how protein function
is controlled by intracellular localization and will serve as a useful
resource for the development of inhibitors that target CRM1-
mediated nuclear export. NESdb may be used to train and test new
NES prediction algorithms to increase the reliability and accuracy of
identifying vague and diverse NESs in the genome.
FIgURE 1: A sample page from NESdb that shows 7 of the 14 illustrated features of the NES from snurportin
1 (SNUPN). The features not shown include full name, alternative names, organism, three-dimensional structures,
comments, references, and a user input form.
We thank Lisa Kinch for insightful suggestions for user interface,
Ming Tang for technical assistance with Web server hosting, and
Maarten Fornerod for discussion. This work is funded by the National
Institutes of Health (F32GM093493 to D.X., R01-GM069909 to
Y.M.C., and R01-GM094575 to N.V.G.), the Welch Foundation (I-1532
to Y.M.C. and I-1505 to N.V.G.), the Leukemia and Lymphoma Soci-
ety Scholar Program (to Y.M.C.), the Cancer Prevention Research
Institute of Texas (PR-101496 to Y.M.C.), and the UT Southwestern
Endowed Scholars Program (to Y.M.C. and N.V.G.).
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