Crystal structure of the PB1 domain of NBR1
Simone Mu ¨ller, Inari Kursula1, Peijian Zou, Matthias Wilmanns*
EMBL-Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany
Received 6 October 2005; revised 7 November 2005; accepted 5 December 2005
Available online 19 December 2005
Edited by Miguel De la Rosa
mission downstream of the serine/protein kinase from the giant
muscle protein titin. Its N-terminal Phox and Bem1p (PB1) do-
main plays a critical role in mediating protein–protein interac-
tions with both titin kinase and with another scaffold protein,
p62. We have determined the crystal structure of the PB1 do-
main of NBR1 at 1.55 A˚resolution. It reveals a type-A PB1 do-
main with two negatively charged residue clusters. We provide a
structural perspective on the involvement of NBR1 in the titin ki-
nase signalling pathway.
? ? 2005 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
The scaffold protein NBR1 is involved in signal trans-
Keywords: PB1 domain; Titin; Signal transduction; X-ray
A modular scaffold domain, named after the prototypical
domains found in Phox and Bem1p (PB1), has been recently
identified to mediate polar, heterodimeric interactions by two
opposite types of PB1 domains, referred as to type-A and
type-B [1,2,17]. Heterodimeric assembly involves specific
electrostatic interactions consisting of a conserved acidic
DX(D/E)GD segment of the so-called OPCA motif  from
an type-A PB1 domain and a conserved lysine residue from
a B-type PB1 domain. A second acidic–basic cluster (A2–B2)
has been detected in the PB1/PB1 interaction of p67phox–
p40phox, aPKCi-par6a, and bem1p-cdc24p [2,4,5]. Additional
specificity is determined by the high sequence diversity in other
parts of the structure, deletions and insertions in loop regions,
and by varying tilt angles [2,4,5].
The cDNA for NBR1 was isolated from an expression li-
brary using the antiserum directed against the ovarian tu-
mour antigen CA125, which is different from NBR1 [6,7].
Despite its location adjacent to the BRCA1 gene, a role in
ovarian or breast cancer could not be demonstrated . Yeast
two-hybrid and co-immunoprecipitation data using in vitro
and in vivo translated proteins revealed a novel interaction
between the PB1 domains of NBR1 and p62 . A recent
analysis of the downstream signalling pathway of the ser-
ine/protein kinase from the giant muscle protein titin revealed
that the PB1 domain NBR1/p62 interaction is an important
connecting link in this pathway, culminating in nuclear tran-
scription . The interaction between titin kinase and the N-
terminus of NBR1 that contains the PB1 domain was
mapped to the first helix from the autoregulatory domain
of the kinase. In an attempt to investigate the molecular com-
ponents involved in this pathway, we have been able to deter-
mine the X-ray structure of the PB1 domain from the scaffold
2. Materials and methods
2.1. Sample preparation
The PB1 domain encoding region of the human NBR1 gene (res-
idues 1–85) was amplified by PCR and cloned into the KpnI–NcoI
sites of a pETM11 vector containing an N-terminal His tag. Protein
expression in Escherichia coli strain BL21(DE3)RIL was induced at
OD600= 0.6 at 37 ?C for 3–3.5 h. The cells were frozen at ?20 ?C
after washing in PBS. Cell pellets were thawed on ice, resuspended
in buffer A (300 mM NaCl, 5 mM imidazole, 25 mM Tris, pH 8.0)
with lysozyme and DNaseI, and then lysed by sonification for
2–3 min. After centrifugation, the supernatant was incubated with
Ni–NTA agarose (Qiagen). The resin was first washed with buffer
A, andthen theproteinwas
A + 400 mM imidazole). The eluted protein was incubated overnight
in the presence of about 20 mM b-mercapoethanol and TEV-prote-
ase for cleavage of the hexa-histidine tag. The protein was concen-
trated and then purified by gel filtration through a Superdex 75
10/30 column equilibrated in 20 mM Bis Tris propane, pH 7.4,
50 mM KCl, and 5 mM DTT. The PB1 domain separated from
the tag as a monomer. The protein sequence was confirmed by
mass spectrometry analysis (EMBL proteomics core facility, Heidel-
berg); the protein itself was concentrated to 9 mg/ml for crystallisa-
Crystals were grown at RT (293 K) in hanging drops containing
equal volumes of protein solution and 0.1 M sodium acetate (pH
4.1–4.4) and 1.6–2.2 M ammonium sulphate. The crystals appear after
1–2 days and belong to the hexagonal space group P6322, with unit cell
dimensions of a = 100.7 A˚and c = 42.2 A˚.
elutedwith bufferB (buffer
2.2. Data collection, phasing, and structure refinement
Cryoprotection of the crystals was performed by soaking them in
reservoir solution that contained 25% glycerol. Crystals were flash-
frozen in the liquid nitrogen stream and data were collected at
100 K. To obtain experimental phases by the anomalous dispersion
method crystals were soaked in 1 M sodium bromide, 0.6 M ammo-
nium sulphate, 20% glycerol, and 0.1 M sodium acetate, pH 4.2. Na-
tive X-ray data were collected on a 165-mm MAR CCD detector
mounted on the synchrotron radiation beamline X13 at EMBL/
*Corresponding author. Fax: +49 40 89902 149.
E-mail address: email@example.com (M. Wilmanns).
1Present address: Division of Molecular Structural Biology, Depart-
ment of Medical Biochemistry and Biophysics, Karolinska Institutet,
Tomtebodava ¨gen 6, 17177 Stockholm, Sweden.
0014-5793/$32.00 ? 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 580 (2006) 341–344
DESY, Hamburg, Germany. A multi wavelength anomalous disper-
sion (MAD) data set was collected on a 165-mm MAR CCD detec-
tor at the synchrotron radiation beamline BW7A at EMBL/DESY,
Hamburg, Germany. The data were processed using the DENZO/
SCALEPACK package . Seven bromide sites were located by
SOLVE . RESOLVE  was used for density modification
and for building an initial model. Arp/wArp  was used to extend
the phases to 1.55 A˚and to build the model at that resolution.
Manual model building was carried out with the program O 
and iterative rounds of refinement with REFMAC5 . The result-
ing statistics are summarised in Table 1.
The atomic coordinates and structure factors have been deposited
with the Protein Data Bank as entry 2BKF.
3.1. Overall structure of the NBR1 PB1 domain
The X-ray structure of the PB1 domain of human NBR1
(residues 1–85) has been determined at 1.55 A˚resolution by
the multiple anomalous dispersion method (Table 1 and
Fig. 1). It reveals the topology of an ubiquitin-like b-grasp
fold, reminiscent of those from previously characterised PB1
domains. Its structural similarity to available type-A PB1 do-
mains (p40phox, aPKCi, cdc24p, and MEK5) is in the range
of 1.1–1.5 A˚upon superposition. A structure-based sequence
alignment is shown in Fig. 2.
The structure comprises two a-helices and a mixed b-sheet,
consisting of five b-strands (Fig. 1A). The OPCA motif is pres-
ent in the PB1 domain and adopts a bba fold. The character-
istic type-A residues are located at positions 50 and 54
(Fig. 1B). However, Asp50 is the only residue that is conserved
in the available NBR1 sequences; in some sequences, the sec-
ond residue of the motif is replaced by a glutamate, maintain-
ing the acidic character of the loop.
Independently from our work, an NMR structure of the
same domain has been deposited in the Protein Data Bank
(1WJ6). The r.m.s. deviation of the NMR structure and our
X-ray structure is 0.83 A˚, allowing basically identical conclu-
sions in terms of functional implications.
3.2. Interaction surface of the NBR1 PB1 domain
The electrostatic potential of the molecular surface of
the NBR1 PB1 domain reflects the acidic residues Asp50,
Glu52, Glu54, and Glu63. (Fig. 1B). These residues provide
the molecular framework for the interaction with the basic
cluster in the type-B PB1 domain of p62 . In contrast,
the opposite face of the NBR1 PB1 domain does not reveal
binding sites (not shown), in agreement with a previous
sequence alignment . This feature also supports the
categorisation of the domain as an exclusive type-A domain,
which is also consistent with previous mutational studies
be involvedin additional
PB1 domains are frequently found in scaffold proteins in-
volved in signal transduction pathways. In particular, the
PB1 domains of the scaffolds NBR1 and p62, which have
been identified as components of the titin kinase downstream
signalling pathway, have been proposed to directly interact
with each other [9,10]. In addition to the N-terminal PB1 do-
mains, both protein components comprise a common ZZ zinc
finger domain, and an ubiquitin-association domain; the
remaining parts of their sequences appear to be unrelated
X-ray data collection and refinement statistics
Data collectionNative Bromide (peak)Bromide (inflection) Bromide (remote)
Unit cell (A˚)
Resolution range (A˚)
Figure of merit
a = 100.7, c = 42.2
a = 100.9, c = 42.6
Resolution range (A˚)
Most favoured region (%)
Disallowed region (%)
Number of atoms
Number of water molecules
r.m.s.d. from ideal bond length (A˚)
r.m.s.d. from ideal bond angles (?)
The values in parentheses refer to the highest resolution shell.
ibIhkl(hkl)iwith I(hkl)ithe observed intensity and ÆI(hkl)æ the mean intensity.
S. Mu ¨ller et al. / FEBS Letters 580 (2006) 341–344
(Fig. 3). These findings raise questions about the nature of
the evidenced NBR1/p62 PB1/PB1 interaction and about its
potential state of equilibrium with homo oligomeric assem-
blies of p62.
Indeed, p62 has been described previously to form PB1
domain mediated homo oligomers of variable sizes. Further-
more, there is evidence that the PB1 domain of p62, in addi-
tion to its association with NBR1, interacts with the PB1
domains of the protein kinases MEK5 and aPKC via the
B-type interface [9,17]. This latter finding raises the question
of whether there is binding competition. Although it is pos-
sible to form a hetero-dimeric type-A/B PB1/PB1 complex
using NBR1/p62 mutants in vitro (, Mu ¨ller et al., unpub-
lished), the dynamics of the proposed interactions under
in vivo conditions still remain to be determined. It is likely
that their formation is spatially and temporally distinct and
may be controlled by other scaffolds or post-translational
In addition, the N-terminus of NBR1, including its PB1
domain, has been reported to interact with the serine/threo-
nine protein kinase from the giant muscle protein titin .
The interaction was found only with a kinase construct in
which part of the C-terminal autoinhibitory segment was
missing (kin3) , suggesting that it is sensitive to its con-
formational state, which is regulated by a peculiar type of
phosphorylation of the P + 1 loop and by the conformation
of its C-terminal autoregulatory segment . However, to
date, the conformation revealed by the kin3 construct of ti-
tin kinase  remains unknown, both under in vitro condi-
tions and under physiological conditions where no such
dynamics simulations has suggested a conformation that
may be connected to mechanical stress . So far, we have
not been able to bind synthetic peptides from the suggested
NBR1 binding site of titin kinase to the NBR1 PB1 domain
(data not shown). This finding suggests that the interaction
may require other parts of NBR1 or may only be present
within the framework of a specific conformation of the over-
In summary, we have been able to determine the structure
of one of the key scaffold proteins – the PB1 domain of
NBR1 – involved in the titin kinase signalling pathway .
The structure will help to unravel key questions about this
Fig. 1. Overall structure of the PB1 domain of NBR1 (1–85). (A)
Ribbon representation. The type-A PB1 domain residues Asp-50, Glu-
52, Glu-54, and Glu-63 are shown in ball-and-stick representation; (B)
Electrostatic potential surface of the NBR1 PB1 domain; red indicates
negatively charged surface areas and blue indicates positively charged
surface areas. The acidic cluster A1 is comprised of Asp50, Glu52, and
Glu54; A2 is comprised of Asp63.
Fig. 2. Structure-based sequence alignment of PB1 domains with known 3D-structures. The structures have been obtained from the PDB: cdc24p
(1Q1O), p40phox, and p67phox (1OEY), MEK5 (1WI0), aPKCi and par6a (1WMH), and bem1p (1IPG). The secondary structural elements, as
observed in the X-ray structure of the PB1 domain from NBR1, are shown above the alignment. The residues constituting the OPCA motif are boxed
with grey background. The type-A and type-B recognition motifs are shown in red and blue, respectively, and the corresponding sequence codes are
coloured accordingly (type-A/B, green).
S. Mu ¨ller et al. / FEBS Letters 580 (2006) 341–344
Acknowledgements: We thank Dr. Andrey Yakovenko and Prof. Download full-text
Mathias Gautel for cDNA of the N-terminal part (residues 1–199) of
the nbr1 gene. Santosh Panjikar is thanked for advice during X-ray
crystallography experiments. The work has been supported by the Re-
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Fig. 3. Model of the TK/NBR1/P62 interaction. The domains with available high resolution structures are shown as ribbons, the remaining domains
are shown schematically. The ribbon of titin kinase has been created from the PDB entry 1TK1 . The sequence segments suggested to be involved
in the interaction of the p62/NBR1 and NBR1/titin kinase haven been taken from  and , respectively. To date, there is no detailed analysis
available, mapping homo association sites in NBR1. We hypothesise an interaction via the central coiled-coil regions. The first helix of the
autoregulatory segment of titin kinase that is involved in the interaction with NBR1  is shown in red. Please note that there is no experimental
structure of the ‘kin3’ conformation of titin kinase  yet. The N-terminal sequence of titin is indicated by a dashed line, reflecting the giant length
of the sequence.
S. Mu ¨ller et al. / FEBS Letters 580 (2006) 341–344