Biomol NMR Assign (2015) 9:223–227 DOI 10.1007/s12104-014-9579-6
ARTICLE
Solid-state NMR resonance assignments of the filament-forming CARD domain of the innate immunity signaling protein MAVS Lichun He • Thorsten Lu¨hrs • Christiane Ritter
Received: 14 July 2014 / Accepted: 26 September 2014 / Published online: 10 October 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The mitochondrial antiviral signalling protein (MAVS) is a central signal transduction hub in the innate immune response against viral infections. Viral RNA present in the cytoplasm is detected by retinoic acid inducible gene I like receptors, which then activate MAVS via heterotypic interactions between their respective caspase activation and recruitment domains (CARD). This leads to the formation of active, high molecular weight MAVS complexes formed by homotypic interactions between the single N-terminal CARDs of MAVS. Filaments formed by the N-terminal MAVSCARD alone are sufficient to induce the autocatalytic conversion from a monomeric to an aggregated state in a prion-like manner. Here, we present the nearly complete spectroscopic 13C and 15N resonance assignments of human MAVSCARD filaments obtained from a single sample by magic angle spinning solid-state NMR spectroscopy. The corresponding secondary chemical shifts suggest that the filamentous form of MAVSCARD retains an exclusively alpha-helical fold that is very similar to the X-ray structure determined previously from monomeric MAVSCARD-maltose binding protein fusion constructs. Keywords MAVS CARD Fibrils Solid-state NMR Assignments Secondary structure
Electronic supplementary material The online version of this article (doi:10.1007/s12104-014-9579-6) contains supplementary material, which is available to authorized users. L. He T. Lu¨hrs C. Ritter (&) Laboratory of Macromolecular Interactions, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany e-mail:
[email protected]
Biological context Upon recognition of viral nucleic acids, retinoic acid inducible gene I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) activate their common downstream adapter protein mitochondrial antiviral signalling protein (MAVS), which is also named virus-induced signaling adapter (VISA), or caspase activation and recruitment domains (CARD) adapter inducing IFN-beta (CARDIF) (Kawai et al. 2005; Meylan et al. 2005; Seth et al. 2005; Xu et al. 2005), through heterotypic CARD– CARD interactions. This interaction results in the assembly of MAVS into high molecular weight, filamentous complexes (Hou et al. 2011; Tang and Wang 2009). The assembled MAVS then serves as a scaffold mediating its interaction with tumor necrosis factor receptor-associated factors (TRAF2, TRAF3, TRAF5 and TRAF6), receptor interacting protein 1 (RIP1) and Fas-associated death domain (FADD) (Kawai and Akira 2009; Liu et al. 2011; Meylan et al. 2005; Michallet et al. 2008; Seth et al. 2006). This orchestration leads to the activation of interferon regulatory factor 3 (IRF-3) and nuclear factor jB (NF-jB). These two factors then move into the nucleus to initiate the expression of type I interferon and other inflammatory cytokines. Notably, filaments formed by the N-terminal MAVSCARD alone could also induce inactive full-length MAVS to assemble into filaments and to rapidly amplify the immune signalling in a prion-like manner (Hou et al. 2011). To better understand the structural basis for MAVS activation, atomic resolution structural information on the MAVSCARD filament would be required. In general, filamentous proteins are incompatible with the two major established techniques in structural biology, X-ray crystallography and solution NMR. However, solid-state NMR has recently become a powerful new technique for the
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determination of atomic-resolution protein structures, and regularly ordered filamentous proteins are ideal candidates for this approach (Habenstein et al. 2012; Loquet et al. 2012; Ritter et al. 2005; Shahid et al. 2012; Van Melckebeke et al. 2010). Here, we present the nearly complete assignment of 13C and 15N chemical shifts of filamentous MAVSCARD, as well as the determination of secondary structural elements of filamentous MAVSCARD from the Ca and Cb chemical shifts. The data was obtained from a single sample of uniformly 13C, 15N labelled MAVSCARD filaments. A set of 2D and 3D experiments was carried out to achieve the backbone and side chain 13C and 15N chemical shift assignments. This assignment data will be helpful for further structure calculation, dynamic investigation or interaction studies with its transducers.
Methods and experiments Sample preparation The sequence of human MAVSCARD (residues 1–100) was obtained from NCBI database. The coding regions were optimized and synthesized by Geneart/Life Technologies Inc. Both genes were cloned into modified pET21-d vector with an N-terminal hexahistidine tag and SUMO* upstream of the initiation codon of MAVSCARD. The plasmid was transformed into T7 express competent E. coli (New England Biolabs) for protein expression. Uniformly 15 N/13C labeled protein was prepared by growing cells in CN-040 minimal medium containing 1 g/l 15NH4Cl and 4 g/l U-13C glucose at 37 °C (Hellert et al. 2013). When the OD600nm of the cell culture reached 1.0, the cells were transferred to a 30 °C incubator and protein expression was induced by 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for 5 h. Cells were then harvested by centrifugation at 5,000g for 20 min. The pellet was resuspended in 50 ml 20 mM TRIS, 50 mM NaCl, 1 mM DTT pH 8.0 buffer with 0.1 mg/ml lysozyme and 0.01 mg/ml DNase. Efficient cell lysis was performed by using a homoginizer (Avestin) for two cycles at 20 kpsi at 4 °C. The soluble bacterial lysate was separated from cell debris and other components by centrifugation at 16,000g for 45 min. Ultracentrifugation was then applied to the supernatant at 100,000g for 2 h. Self-assembled MAVSCARD was collected in the pellet. This pellet was resuspended again in 20 mM TRIS, 50 mM NaCl, 2 mM DTT pH 8.0 buffer and simultaneously cleaved with SUMO* protease at 4 °C overnight. The sample was loaded onto the Ni–NTA column (Qiagen). His-tagged SUMO* bound to the column, while the flow-through contained MAVSCARD filaments, which were further purified via a Superdex 200 column (GE Healthcare) using 20 mM TRIS, 50 mM NaCl, 1 mM DTT pH
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8.0 buffer to remove lipids and other contaminants. The sample was packed into a 3.2 mm thin-wall NMR rotor by centrifugation at 100,000g for 15 h using a specially designed filling device (Gardiennet et al. 2012). NMR spectroscopy All spectra were recorded on a Bruker Avance III 600 MHz spectrometer operating at a static field of 14.1 T equipped with a 3.2 mm Bruker triple-resonance MAS probe. The spectra were recorded at a sample temperature of 5 ± 1 °C. The pulse sequences were implemented as reported recently (Shi and Ladizhansky 2012). 15N and 13C transfer was established through band-selective crosspolarization (Baldus et al.1998) and SPINAL64 decoupling of 90 kHz (Fung et al. 2000) was used during direct and indirect chemical shift evolution. Further details of the experimental setups are given in Table S1. All spectra were processed using Topspin 3.2 (Bruker Biospin) by apodizing with a cosine square function, zero filling to a power of 2, closing to two times of the number of points measured. Spectral analysis, peak picking, and assignments were performed using the CcpNmr software package (Fogh et al. 2002; Stevens et al. 2011; Vranken et al. 2005). Sequential backbone 13C and 15N resonance assignments were achieved using a suite of 3D experiments, namely NCACX, CANCO, NCOCX, where CX denotes any carbon, and 15 ms DARR experiments. First, the 2D DARR spectrum was analyzed to identify ‘fingerprints’ of various types of amino acids. For the sequence-specific assignment of the residues, the 15N[i] and 13Ca[i] chemical shifts of each amino acid were identified from the 3D NCACX spectrum. Next, these chemical shifts were linked with the 13C0 [i - 1] with the help of the CANCO spectrum. Then, the NCOCX spectrum was used to assign the chemical shifts of 13Ca[i - 1], 13Cb[i - 1] and parts of the side chains. Complete side chain resonance assignment was achieved using the longer mixing time NCACX (300 ms) and DARR spectra (100 ms/250 ms/400 ms). In addition, the 300 ms NCACX spectrum was also used to confirm the assignment, because magnetization exchange between 13 Ca[i] and 13Ca[i - 1] was observable.
Assignment and data deposition MAVSCARD was purified in a filamentous state from E. coli (Fig. S1). The filaments consist of 102 residues, of which the first two residues are glycine and serine remaining after cleavage of the SUMO* tag. The filament preparation of MAVSCARD gives highly resolved resonances. Figure S2 shows a 1D 13C spectrum of MAVSCARD along with its sequence. It is evident that the sample attained a high
Solid-state NMR resonance assignments
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Fig. 1 An example of a sequential assignment walk for residues 82–87 of MAVSCARD. Strips plot extracted from 3D CONCA, NCACX and NCOCX experiments illustrate the sequential
assignment strategy. The NCACX spectrum is shown in green. The NOCX spectrum is shown in black and the CONCA spectrum is shown in cyan
degree of microscopic order and homogeneity, leading to narrow line widths. The highly ordered state and homogeneity of MAVSCARD fibers enabled us to collect high resolution 2D and 3D correlation spectra with excellent signal to noise ratio within reasonable times (Table S1). The sequential backbone assignments and the side chain assignments were obtained from a single uniformly labeled sample. Three 3D chemical shift correlation experiments (CONCA, NCACX and NCOCX) and one 2D DARR experiment (mixing time 15 ms) were acquired for this purpose. To illustrate sequential assignments, a representative strip plot of NCACO, NCACX and NCOCX is shown in Fig. 1. The observed peak width in the spectra is between 0.5 and 1 ppm and there was almost no signal overlap in the CANCO spectrum, allowing the nearly complete resonance assignment using the afore-mentioned strategy. To confirm this assignment, a 300 ms NCACX spectrum was analyzed to check the self-consistency of the sequential assignment. It provided cross peaks between two sequential residues (Fig. S3), as 13Ca not only exchanged with the side chain carbons of the same residue but also with the backbone and side-chain spins of the previous residue.
Excluding the first two residues GS remaining after cleavage of the SUMO* tag, 99 % of the Ca, 97 % of the Cb and 96 % of the carbonyl carbon resonances could be assigned. The backbone amide nitrogen chemical shifts were assigned for residues 4–97 (Table S2). The chemical shifts have been deposited to BioMagResBank (http:// www.bmrb.wisc.edu) as BMRB entry 25076. Figure 2 shows a 15 ms DARR (Takegoshi et al. 2001) 13C–13C spectrum, which displayed only little spectral overlap in both the aliphatic and carbonyl regions. The entire set of cross peaks could be explained by the assigned chemical shifts. A region spanning from residues R37 to R41, was only weakly visible in the CONCA spectrum, although not impeding the sequential walk. However, it might suggest a more dynamic behavior in this region compared to the rest of the protein. Given the fact that almost all backbone residues could be faithfully assigned, it appears that there are no major flexible loop regions in the MAVSCARD protomers. Based on the sequence of the protein, the backbone 13C chemical shifts were used for experimental prediction of the secondary structure using TALOS? (Shen et al. 2009). More than three positive values in a row indicate a-helical
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δ 2 - 13 C / ppm
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89A 44A 27I 85A 74A 77A 73I 36A 24V8T 45T 69V 4A 61T 91V 35T 22V 25V 47T 82V 88V 84L 24V 54T 22V 58L 25V 80E 31L 46C 39Q 88V 80E 32P 48L 28L 55L 77R 93Q 20C 81L 79C 70E 43R 34L13C 29P 5E 63Q 33C 70E 68W 64R 87E 7K 32P 87E 57H 88V 22V 96Q 91V 39Q 27I 24V 14R 93Q 87E 26E18N 80E 70E 12I 87E 73I 21N 80E 71Y 30Y 86D 81L 26E 15N 5E 19F 86D 72F 40D 58L 76L 51N 50G 77R 84L -2G 42L 23D 62L 23D 48L 78G 55L 67G 51N 21N15N 31L 32P 63Q 23D 15N 23D 89A 81L 65R 96Q 44A 34L 4A 18N 33C 85A 74A 64R 40D 60N 36A 14R 53D 62L 68W 86D 30Y 6D 19F 49S 42L 93Q 87E 79C 58L 51N 94S 70E 35T 37R 20C 77R 92Y 39Q 22V 17S 94S 71Y 32P 27I 56W 66P 90S 29P 49S 16F 47T 91V 45T 54T 12I 54T 47T 82V 45T 88V 25V 73I 8T 61T 69V 8T 35T 24V
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45T 4A 91V 35T 69V 8T 24V 45T 55L 77A 58L 82V 22V 35T 42L 61T 34L 22V 28L 76L 48L 81L 24V 31L 10K 46C 47T 88V 62L 7K 81L 13C 39Q 48L 63Q 34L 54T 79C 32P 66P 81L 32P 88V 54T 42L 43R 29P 66P 62L 41R 69V 77R 64R 29P 29P 93Q 73I 96Q 34L 31L 56W 70E 68W 25V 87E 29P 14R 63Q 37R 88V 32P 26E 33C 41R 24V 82V 32P 10K 43R 39Q 22V 66P 66P 70E 80E 18N 30Y 9Y 87E 5E 21N 12I 19F 59F 26E 11Y 83D 72F 15N 60N 71Y
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42L 55L 34L 76L 58L 22V 28L 22V 48L 55L 81L 34L 25V 24V 58L 31L 80E 31L 25V 76L 58L 76L 62L 28L 39Q 88V 10K 48L 10K 81L 12I 66P 48L 63Q 34L 58L 84L 93Q 76L 42L 81L 64R 32P 81L 62L 34L 77R 96Q 10K 5E 43R 31L 70E 87E 52R 73I 64R 64R 34L 43R 26E 43R 41R 98R 27I 14R 7K
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Fig. 2 Contour plot of 2D 13C–13C DARR spectrum. 2D DARR experiment of MAVSCARD with 15 ms mixing time. The aliphatic region and carbonyl region are shown together with chemical shift
Fig. 3 Chemical shift index (CSI) analysis. Secondary chemical shift difference plotted as a function of residue number. Three or more positive shifts in a row indicates a helix secondary structure. Schematic representation of helical segments predicted from CSI are given below the plot in cyan. Helical segments observed in the crystal structure of monomeric MAVSCARD are given in grey
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assignments, which were achieved by analysis of 3D heteronuclear NCACX, NCOCX and CONCA spectra
Secondary structure of crystal structure
Solid-state NMR resonance assignments
conformation, three or more negative ones indicate a bstrand (Wishart and Sykes 1994). Figure 3 displays the secondary chemical shifts of the assigned residues. Most of the assigned residues are found in a-helical conformation as depicted by the representative schematic below the plot. Clearly, the chemical shift index (CSI) indicates the existence of six helices separated by five short turns, in addition to the two terminal stretches. Only minor deviations in the length of the secondary structure elements relative to the monomeric form, determined by X-ray crystallography, can be observed (Fig. 3). It appears thus plausible that MAVSCARD retains the greek-key topology, six-helix bundle architecture that has been determined for its monomeric conformation. Acknowledgments We are grateful to Dr. Mumdooh Ahmed for recording the ssNMR spectra and for helpful discussions. L. H. has been supported by a fellowship from the HZI graduate school.
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