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. 2007 Sep 4;104(36):14336-41.
doi: 10.1073/pnas.0703012104. Epub 2007 Aug 28.

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

James M Murphy  1 , Dmitry M Korzhnev, Derek F Ceccarelli, Douglas J Briant, Arash Zarrine-Afsar, Frank Sicheri, Lewis E Kay, Tony Pawson
Affiliations

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

James M Murphy et al. Proc Natl Acad Sci U S A. .

Abstract

The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Crystal structure of hMARK3 catalytic and UBA domains. (A) Cartoon of the hMARK3 (residues 48–370) crystal structure. The N- and C- termini are labeled in black text. The kinase domain (AMGS from vector and D48–N308) is colored red; the linker (A309–D328) is yellow; and the UBA domain (Q329–R366) is blue. The dashed red line corresponds to the activation segment residues (205–208) for which no electron density was observed. (B) Comparison of the hMARK3 UBA domain and the canonical UBA domain fold. Structure of hMARK3 UBA domain from hMARK3 (residues 48–370) crystal structure. Residues involved in the hydrophobic core of the UBA domain are depicted by yellow sticks. (C) Structure of HHR23A (1) UBA domain. Residues involved in the hydrophobic core of the UBA domain are depicted by yellow sticks. The hMARK3 UBA domain contains an inversion of the α3 helix compared with this canonical UBA domain topology. Figure was drawn from PDB ID code 1IFY (18).
Fig. 2.
Fig. 2.
NMR spectroscopy characterization of millisecond to microsecond exchange processes within the hMARK3 UBA domain. (A) 1H-15N HSQC spectrum of the hMARK3 UBA domain. HSQC spectrum recorded at 25°C, 500 MHz. The protein sequence is drawn above the spectrum, with assigned residues underlined and helices indicated by arrows above the corresponding sequence. Assignments are annotated next to the corresponding peaks in the spectrum in black text. The boxed region is enlarged in Right Inset. Peaks marked with × were not assigned, and * marks a side chain resonance. G(−1) and S(0) arise from the cloning vector. (B) Representative 15N relaxation-dispersion profile. 15N relaxation-dispersion profile for the backbone amide resonance of E343 derived from the CPMG experiment at 25°C. The upper and lower curves are derived from data collected at 800 and 500 MHz, respectively. (C) Representative 13C relaxation-dispersion profile. 13C-methyl relaxation-dispersion profile for a Leu δ1 or δ2 methyl resonance (assignment unavailable) derived from the CPMG experiment at 5°C. The upper and lower curves are derived from data collected at 800 and 500 MHz, respectively.
Fig. 3.
Fig. 3.
Ub binds the hMARK3 UBA domain via a classical interaction interface. Weighted-average chemical shift perturbation (Δδav) versus residue number. Δδav, calculated as described in Materials and Methods, for each residue in 15N-Ub 1H-15N HSQC spectra in the absence versus presence of 5.3 molar equivalents of unlabeled hMARK3 (residues 320–375). (Inset) The hMARK3 UBA domain binding surface of Ub is spatially clustered around K48. Backbone nitrogen atoms of Ub residues exhibiting Δδav > 0.05 ppm are drawn as spheres on the structure of Ub (PDB ID code 1UBQ). This UBA domain binding surface is analogous to that implicated in the canonical Ub interaction with the hHR23A UBA domains (21).
Fig. 4.
Fig. 4.
A reciprocal NMR titration confirms that the hMARK3 UBA domain binds monoUb. (A) Δδav versus residue number for hMARK3 UBA domain in the presence of monoUb. Δδav was calculated for each residue in 1H-15N HSQC spectra in the absence versus presence of 5.8 molar equivalents of monoUb. No data, and thus no bars in this chart, were available for peaks absent from 2D spectra. (Inset) Ub-interacting residues of the hMARK3 UBA domain. Backbone nitrogen atoms corresponding to residues exhibiting Δδav > 0.05 ppm are indicated as spheres on the structure of the hMARK3 UBA domain from the kinase:UBA domain crystal structure (Fig. 1). The UBA domain is rotated 180° about the x axis compared with the orientation in Fig. 1B. The backbone N spheres of G339 and Y340 of the MGY motif are labeled in black text. (B) Characteristic plots of Δδ1H versus Ub/UBA molar ratio. Plot of absolute 1H chemical shift change as a function of addition of ligand for V336 (filled diamonds) and G339 (open circles). The binding isotherm for Q342, also used in the calculation of Kd, was omitted for clarity because this curve overlays with that of V336. Nonlinear curve-fitting was used to determine the Kd for the hMARK3 UBA domain interaction with monoUb, as described in Materials and Methods.
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