Article

Crystal structure of the PB1 domain of NBR1

EMBL-Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
FEBS Letters (Impact Factor: 3.34). 02/2006; 580(1):341-4. DOI: 10.1016/j.febslet.2005.12.021
Source: PubMed

ABSTRACT The scaffold protein NBR1 is involved in signal transmission downstream of the serine/protein kinase from the giant muscle protein titin. Its N-terminal Phox and Bem1p (PB1) domain plays a critical role in mediating protein-protein interactions with both titin kinase and with another scaffold protein, p62. We have determined the crystal structure of the PB1 domain of NBR1 at 1.55A resolution. It reveals a type-A PB1 domain with two negatively charged residue clusters. We provide a structural perspective on the involvement of NBR1 in the titin kinase signalling pathway.

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    • "A series of very recent studies has provided valuable information on the structural details that govern binding between the different PB1 modules (Hirano et al., 2004; Ito et al., 2001; Leitner et al., 2005; Noda et al., 2003; Terasawa et al., 2001; Wilson et al., 2003; Yoshinaga et al., 2003) and explains how they direct the formation of different macromolecular signaling complexes. Crystallographic and NMR experiments have established the 3D structure of a number of PB1 domains (Hirano et al., 2004, 2005; Muller et al., 2006; Terasawa et al., 2001; Wilson et al., 2003; Yoshinaga et al., 2003). All of them display the topology of a ubiquitin-like b-grasp fold, including six-stranded b-sheets and two a helices (Figure 2A). "
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    ABSTRACT: The PB1-domain-containing proteins p62, aPKC, MEKK2/MEKK3, MEK5, and Par-6 play roles in critical cell processes like osteoclastogenesis, angiogenesis, and early cardiovascular development or cell polarity. PB1 domains are scaffold modules that adopt the topology of ubiquitin-like beta-grasp folds that interact with each other in a front-to-back mode to arrange heterodimers or homo-oligomers. The different PB1 domain adaptors provide specificity for PB1 kinases to ensure the effective transmission of cellular signals. Also, recent data suggest that PB1 domains may serve to orchestrate signaling cascades not involving other PB1 domains, such as the MEK5-ERK5 and p62-ERK1 interactions.
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    ABSTRACT: The giant muscle protein titin is the largest polypeptide known and constitutes, in addition to actin and myosin, the third filament system in the striated muscle sarcomere. Titin spans half of the sarcomere and interacts with many proteins along all its range. Three regions of accumulated interaction and associated signalling are found in the Z-disc, the I-band and the M-line, respectively. Among about 300 predicted domains in titin, to date only one has been identified to comprise a catalytic function, a serine/threonine kinase domain within the M-line, referred to as ’titin kinase’. The aim of this work was to unravel the structural, molecular context of titin kinase in terms of adjacent titin domains and downstream signalling domains. The tandem immunoglobulin domains A168-A169 are located amino-terminal to titin kinase at the end of the A-band within titin in the sarcomere. The structure solved in this work implies that these two domains are tightly connected via a continuous β-strand by merging of the last β-strand of A168 with the first of A169. A bulge is formed between two strands in A169 at a position which is rather uncommon for an insertion in an immunoglobulin (Ig) domain. This insertion is involved in the interaction of A168-A169 with muscle specific RING protein MURF-1. In addition to A168-A169, the carboxy-terminal domain to titin kinase, the Ig domain M1 was solved. The proteins NBR1 and p62 are substrates of titin kinase, and NBR1 links p62 to titin kinase. The interaction of NBR1 and p62 within the signalling pathway was studied here. Both proteins interact via their N-terminal PB1 domain, a recently identified interaction domain. The structure of NBR1 PB1 was solved as a single domain and in complex with p62 PB1. The complex reveals two patches of positive (p62 PB1) and negative (NBR1 PB1) charge. The affinity of the complex is in the nanomolar range. This work extends our structural knowledge about titin immunoglobulin domains. Furthermore, it contributes to the understanding of interaction among domains of the titin kinase downstream signalling pathway. Thereby, it may help to unravel the connection between signalling related to titin stretching and transcription control in the context of muscle degradation. Das Muskelprotein Titin ist das größte bekannte Polypeptid und stellt neben Aktin und Myosin das dritte Filamentsystem im Sarkomer des gestreiften Muskels dar. Titin erstreckt sich über die Hälfte des Sarkomers und interagiert mit vielen Proteinen entlang seiner Spannweite. Drei Regionen mit gehäuft vorkommenden Interaktionen und damit verbundener Signalübertragung befinden sich in der Z-Scheibe, der I-Bande und der M-Linie. Unter den etwa 300 vorhergesagten Domänen in Titin, wurde bis heute nur eine mit katalytischer Funktion identifiziert, eine Serine/Threonin Kinase Domäne in der M-Linie, auch als ”Titin Kinase” bezeichnet. Das Ziel dieser Arbeit war es, den strukturellen, molekularen Zusammenhang von Titin Kinase bezüglich benachbarter Titin Domänen und nachgeschalteter Signaldomänen aufzudecken. Die Tandem Immunoglobulin Domänen A168-A169 befinden sich N-terminal zu Titin Kinase am Ende der A-Bande in Titin im Sarkomer. Die in dieser Arbeit gelöste Struktur impliziert, dass diese Domänen über ein durchgehendes β-Faltblatt verknüpft sind, durch Verschmelzung des letzten β-Faltblattes von A168 mit dem ersten β-Faltblatt von A169. An einer für eine Immunoglobulin- (Ig) Domäne eher ungewöhnlichen Stelle ist eine Auswölbung zwischen zwei Faltblättern in A169 ausgebildet. Diese Insertion ist an der Interaktion von A168-A169 mit dem muskel-spezifischen RING Protein MURF-1 beteiligt. Zusätzlich zu A168-A169 wurde die sich C-terminal zu Titin Kinase befindende Domäne, die Ig Domäne M1, gelöst. Die Proteine NBR1 und p62 sind Substrate von Titin Kinase, und NBR1 verbindet p62 mit Titin Kinase. Hier wurde die Interaktion von NBR1 und p62 im Signalweg untersucht. Beide Proteine interagieren über ihre N-terminale PB1 Domäne, eine erst kürzlich identifizierte Domäne. Die Strukturen von NBR1 PB1 als einzelne Domäne sowie im Komplex mit p62 PB1 wurden gelöst. Der Komplex lässt jeweils zwei Stellen positiver (p62 PB1) und negativer (NBR1 PB1) Ladung erkennen. Die Affinität des Komplexes befindet sich im nanomolaren Bereich. Die vorliegende Arbeit erweitert damit unser strukturelles Wissen über Immunoglobulin Domänen von Titin. Darüber hinaus trägt sie zum Verständnis von Interaktionen zwischen Domänen des Titin Kinase Signalweges bei. Dies könnte helfen, die Beziehung von Signalen, die durch Dehnung von Titin ausgelöst werden, und Transkriptionskontrolle im Zusammenhang von Muskelabbau aufzudecken.
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