Structural Variations in the Catalytic and Ubiquitin-associated Domains of Microtubule-associated Protein/Microtubule Affinity Regulating Kinase (MARK) 1 and MARK2

Alexander Marx,Chanakya Nugoor,Jens Müller,Saravanan Panneerselvam,Thomas Timm,Matthias Bilang,Efstratios Mylonas,Dmitri I Svergun,Eva-Maria Mandelkow,Eckhard Mandelkow

doi:10.1074/jbc.m604865200

Abstract

The microtubule-associated protein (MAP)/microtubule affinity regulating kinase (MARK)/Par-1 phosphorylates microtubule-associated proteins tau, MAP2, and MAP4 and is involved in the regulation of microtubule-based transport. Par-1, a homologue of MARK in Drosophila and Caenorhabditis elegans, is essential for the development of embryonic polarity. Four isoforms of MARK are found in humans. Recently, we reported the crystal structure of the catalytic and ubiquitin-associated domains of MARK2, an isoform enriched in brain (Panneerselvam, S., Marx, A., Mandelkow, E.-M., and Mandelkow, E. (2006) Structure 14, 173-183). It showed that the ubiquitin-associated domain (UBA) domain has an unusual fold and binds to the N-terminal lobe of the catalytic domain. This is at variance with a previous low resolution structure derived from small angle solution scattering (Jaleel, M., Villa, F., Deak, M., Toth, R., Prescott, A. R., Van Aalten, D. M., and Alessi, D. R. (2006) Biochem. J. 394, 545-555), which predicts binding of the UBA domain to the larger, C-terminal lobe. Here we report the crystal structure of the catalytic and UBA domain of another isoform, MARK1. Although the crystal packing of the two isoforms are unrelated, the overall conformations of the molecules are similar. Notably, the UBA domain has the same unusual conformation as in MARK2, and it binds at the same site. Remarkable differences occur in the catalytic domain at helix C, the catalytic loop, and the activation segment.

Highlights

Bule binding repeats [1]
The ubiquitin-associated domain (UBA) domain has the same fold as observed for MARK2, with the third helix pointing in the opposite direction compared with UBA domains of other proteins
The UBA domain of MARK1 binds at the same site as in MARK2, at the N-terminal lobe (N-lobe) and on the back side of the active site

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation and Crystallization—Fragments of MARK1 from human (GenBankTM accession number Q9P0L2) were cloned and expressed in Escherichia coli strain BL21 AI (Invitrogen) by using the manufacturer’s protocol. The MARK1 fragment Asn-45—Lys-371 described here is similar to the MARK2 fragment Asn-39 —Lys-364 [17], which was identified by limited proteolysis. MARK1 has a single, one-residue insert (Thr-51) relative to MARK2. Residue ranges Asn-45—Ala-50 and Asp-52—Lys-371 of MARK1 correspond to Asn-39 —Ala-44 and Asp-45—Lys-364 in MARK2. In the following we use the residue numbers based on the MARK2 sequence for both isoforms. All proteins were prepared as described [9, 17]. Crystals of MARK1 were grown by vapor diffusion by mixing 2 ␮l of protein (20 mg/ml) with 2 ␮l of a reservoir solution containing 7–10% polyethylene glycol 3350, 0.1 M Bis-Tris pH 6.5, 0.2 M ammonium citrate at 4 °C. For SAXS analysis 1 mM dithiothreitol (DTT) was added during the purification procedure, and SEPTEMBER 15, 2006 VOLUME 281 NUMBER 37

Data collection

RESULTS

DISCUSSION