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. 2011 Jun 24;409(5):722-41.
doi: 10.1016/j.jmb.2011.04.018. Epub 2011 Apr 21.

Structural features and chaperone activity of the NudC protein family

Meiying Zheng  1 , Tomasz Cierpicki, Alexander J Burdette, Darkhan Utepbergenov, Paweł Ł Janczyk, Urszula Derewenda, P Todd Stukenberg, Kim A Caldwell, Zygmunt S Derewenda
Affiliations

Structural features and chaperone activity of the NudC protein family

Meiying Zheng et al. J Mol Biol. .

Abstract

The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.

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Figures

Figure 1
Figure 1
The molecular architecture of the proteins in the NudC family. The CS-domain is shown in dark grey. The C-terminal black α-helices and black intrinsically disordered fragment at the N-terminus are the regions of highest amino acid conservation within the NudC and NudCL subfamilies. The two putative β-propeller sections (hashed) shown for NudCD1, are likely to constitute two halves of a split domain.
Figure 2
Figure 2
NMR 2D HSQC spectra recorded for three different variants of hNudC: (A), hNudC143–331; (B) hNudC158–331; (C) hNudC158–274.
Figure 3
Figure 3
A comparison of the crystal structure of the CS domain from hNudC, residues 158–274 (shown in A, this work, PDB code 3QOR) with those of (B) Sgt1 (PDB code 2XCM); (C) p23 (PDB code 1EJF); (D) α-crystallin (PDB code 2WJ7); (E) rat Hsp20 (PDB code 2WJ5)
Figure 4
Figure 4
The structures of CS domains from various NudC family proteins; in all figures the core β-strands are shown in read, and the N-terminal strand in yellow: (A) The crystal structure of the CS domain from E. cuniculi (PDB entry 2O30; this structure was determined at 1.7 Å and refined to R/Rfree of 0.217 and 0.256 respectively); (B) NMR structure (best out of 20 conformers) of the 176–286 fragment of the CS domain from human NudCL (PDB 1WGV); (C) The crystal structure of the full length mouse NudCL2 (PDB 2RH0; resolution 1.95 Å, R/Rfree 0.192/0.235) – only one ‘monomer’ is shown; the red and green fragments originate from two different molecules in the asymmetric unit. (D) Complete, domain-swapped homodimer of NudCL2; the two molecules are depicted in red and green, respectively.
Figure 5
Figure 5
The principle crystal contacts in the NudCL2 structure showing the assembly of the homodimers into a filament via a conserved hydrophobic surface. The side chains of Trp113, Phe127, Phe134, and Phe136 are shown in detail, but not labeled.
Figure 6
Figure 6
Dimerization is required for the chaperone activity of NudC. (A) Luciferase (0.1 μM, filled squares) was incubated at 42° C for 20 minutes in the presence and absence of 0.8 μM NudC (open circles), construct 1–274 (filled triangles), construct 158–331 (open diamonds), and construct 1–141 (open inverted triangles). Aggregation was measured by light scattering at 370 nm. Data represents the average of 3 independent trials and are expressed as a percentage of maximum aggregation of luciferase at 20 minutes with ± mean standard deviation. (B) CS (0.15 μM, filled squares) was incubated at 44 °C for 20 minutes in the presence and absence of 1.2 μM NudC (open circles), construct 1–274 (filled triangles), construct 158–331(open diamonds), and construct 1–141 (open inverted triangles). Aggregation was measured and calculated as described in A. All NudC concentrations were based off the monomeric form.
Figure 7
Figure 7
Suppression of luciferase aggregation by NudC proteins. (A) Luciferase (0.1 μM, filled squares) was incubated at 42°C in the presence and absence of 0.8 μM NudC (open circles), NudCL (filled triangles), NudCL2 (closed diamonds) and the negative control, NudC construct 158–331 (open diamonds). Data represent the average of 3 independent trials and are expressed as a percentage of maximum aggregation of luciferase at 20 minutes with ± mean standard deviation. (B) Lanes 1,3, and 5 represent the soluble fractions. Lanes 2, 4, and 6 are the insoluble fractions. Lanes 1 and 2 consist of 0.82 μM luciferase by itself. Lanes 3 and 4 consist of 6.56 μM NudCL2 with 0.82 μM luciferase. Lanes 5 and 6 represent the negative control, 6.56 μM NudC construct 158–331 and 0.82 μM luciferase. (c) Lanes 1,3 and 5 represent the soluble fractions and lanes 2,4, and 6 represent the insoluble fractions. Lanes 1 and 2 consist of 0.82 μM luciferase only. Lanes 3 and 4 include 6.56 μM NudCL with 0.82 μM luciferase. Lanes 5 and 6 contain 6.56 μM NudC construct 158–331 and 0.82 μM luciferase. The asterisk represents a degradation product of NudCL. All NudC concentrations assume a monomeric state.
Figure 8
Figure 8
Suppression of CS aggregation by NudC proteins. (A) CS (0.15 μM, filled squares) was incubated at 44 °C in the presence and absence of 1.2 μM NudC (open circles), NudCL2 (closed diamonds), NudCL (filled triangles), and the negative control NudC construct 158–331 (open diamonds). Data represent the average of 3 independent trials and are expressed as a percentage of maximum aggregation of citrate synthase at 20 minutes with ± mean standard deviation. (B) Lanes 1, 3, and 5 represent the soluble fractions. Lanes 2, 4, and 6 are the insoluble fractions. Lanes 1 and 2 consist of 1.6 μM citrate synthase by itself. Lanes 3 and 4 consist of 12.8 μM NudCL2 with 1.6 μM citrate synthase. Lanes 5 and 6 represent the negative control, 12.8 μM NudC construct 158–331 and 1.6 μM citrate synthase. (C) Lanes 1,3 and 5 represent the soluble fractions and lanes 2,4, and 6 represent the insoluble fractions. Lanes 1 and 2 consist of 1.6 uM citrate synthase only. Lanes 3 and 4 include 12.8 μM NudCL with 1.6 μM citrate synthase. Lanes 5 and 6 contain 12.8 μM NudC construct 158–331 and 1.6 μM citrate synthase. All NudC concentrations assumed a monomeric state.
Figure 9
Figure 9
The NudC proteins do not form binary complexes with Lis1. (A) Raw calorimetric titration data for the thee NudC proteins titrated against Lis1 (see methods), showing no enthalpy change. (B) Positive control showing the titration curve of Lis1 against Ndel1, a known partner of Lis1. An ITC profile of the interaction of Ndel1 with Lis1 is shown in upper panel and individual dissipated heats plotted against the molar ratio of interacting proteins in the lower panel. The data were fitted with “one set of sites model” shown as a solid line on lower panel. The best fit resulted in Ka=6.4×105 M−1, n=0.83 and ΔH=−9.5 kcal/mol of dimer. (C) Pull-down experiments showing binary interaction of Lis1 with Ndel1 (see methods), and no interaction with any of the NudC proteins. I, input; U, unbound, i.e. flowthrough; B, bound.
Figure 10
Figure 10
Sequence conservation calculated as described in Methods was mapped onto (A) hNudC, (B) mNudCL, and (C) mNudCL2 (dimer) protein surfaces. The level of conservation is depicted on a colour scale - from blue (least conserved) to red (most conserved). The mNudCL structure and chain A of mNudCL2 were aligned to hNudC and rotated as indicated. The only fully invariant amino acid, based on sequence alignment and topology, is a tryptophan, in each case indicated by an arrow.
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