Backbone Assignment of the MALT1 Paracaspase by Solution NMR

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs). ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the 15N/13C/1H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3) domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS) based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins.


Introduction
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) has a central role in transcription factor NF-κB signalling [1,2]. NF-κB controls the expression of numerous anti-apoptotic and proliferation-promoting genes and has a key role in B-cell activation. Constitutive MALT1 activity is one characteristic of specific types of B-cell lymphomas [3,4], rendering MALT1 as a potential drug target for these malignancies.
MALT1 exerts its regulating function by two routes. Upon antigen stimulation MALT1 acts as a scaffold for a protein complex formed with CARMA1 and Bcl10, the CBM complex [5]. The complex mediates the further events that lead to the nuclear translocation and activation of NF-κB. In addition, MALT1 functions as a protease that acts on several proteins involved in the pathway leading to NF-κB activation [6,7].
MALT1 is the first human paracaspase identified [2]. Differently from caspases, reported MALT1 substrates are all cleaved directly on the C-terminal side of an arginine residue [6,7]. Another difference is that MALT1 is not cleaved upon activation [8]. Similarly to caspases, MALT1 is dependent on dimerization for catalytic activity [8]. In vivo this is accomplished by ubiquitination of a single lysine residue [9]. In biochemical assays it has been found that high concentration of a kosmotropic salt increases MALT1 catalytic activity [7], probably by promoting dimerization.
Full length MALT1 is a 93 kDa protein consisting of 823 amino acids (Uniprot Q9UDY8). The sequence folds into five domains: the N-terminal DEATH domain, two immunoglobulinlike domains (Ig1 and Ig2), the caspase-like domain (Casp) and a third immunoglobulin-like domain (Ig3) followed by an unstructured C-terminal tail. The structures of individual domains and combinations thereof have been solved by X-ray crystallography [8,[10][11][12]. In recent studies MALT1 truncated to the caspase-like and Ig3 domains, MALT1 Casp-Ig3 was used in characterising the structural determinants of activation [8]. The studies revealed that both apo MALT1 Casp-Ig3 (substrate/ligand-free) and MALT1 Casp-Ig3 in complex with the irreversible substrate mimetic ligand (z-VRPR-mono fluoro-ketone) forms dimers. However, the trigger of proteolytic activity is substrate-induced. Activation involves stabilisation of loop regions in the caspase domain by the ligand, resulting in the alignment of the catalytic machinery and folding of the substrate binding pocket. In absence of ligand, the loops are disordered and the substrate binding pocket is collapsed.
The flexibility of the major loops in the caspase domain and their relationship with the Ig3 domain seems central to MALT1 activation. Solution NMR is the method of choice for studying such flexible parts in proteins. As the first step towards NMR spectroscopy elucidation of the structure and dynamics of MALT1 Casp-Ig3 , in solution, . we report, the 15 N/ 13 C/ 1 H backbone assignment of MALT1 Casp-Ig3 in its ligand-free state by high resolution NMR. In addition, we for the first time show that the targeted acquisition (TA) [13][14][15] procedure, which previously has been used only for intrinsically disordered proteins and small globular proteins, perform just as well also for a relatively large 44 kDa protein.

MALT1Casp-Ig3(338-719) expression and purification
The human MALT1 Casp-Ig3 (338-719) construct, consisting of the catalytic domain and the Ig3 domain, was chosen for NMR studies. DNA encoding this sequence and a C-terminal His 6 tag was inserted between the NdeI and XhoI sites in the expression vector pPET21b (Novagen). Expression of the protein was performed in Escherichia coli strain BL21 Star (DE3) (Invitrogen) at 37°C and with 50 μg/ml carbenicillin as the selective antibiotic. For isotope labelling, 15 N/ 13 C/ 2 H-labeled minimal medium based on M9 was prepared in D 2 O. At an OD 600 of approximately 1, induction of the protein expression was started by adding IPTG to 0.4 mM. The cultures were incubated at 18°C for an additional 6 hours. The cells were harvested by centrifugation at 6000g for 20 min and subsequently re-suspended and lysed by high pressure (1,7 kbar) in a Cell Disruptor (Constant Systems). The lysate cleared by centrifugation at 48000g.
The protein was subsequently purified by 3 chromatographic steps. First, an Immobilized metal ion affinity chromatography (IMAC) step on a Ni 2+ Sepharose 6 Fast Flow column (GE Healthcare) was performed. Second, Ion-exchange chromatography (IEX) on a Q-Sepharose HP column (GE Healthcare) was carried out. Third, a size exclusion chromatography (SEC) step using a HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare) was performed. The buffer was subsequently exchanged to a buffer suitable for NMR experiments on Bio-Spin P-6 gel columns (BioRad). Final yield from a culture of 8 litres was approximately 10 mg of purified protein after concentration to 0.5 mM. analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.
Competing Interests: Sofia Unnerståle, Vera Baraznenok, Gun Stenberg, Jimmy Lindberg and Tatiana Agback are employed by Medivir AB. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
To enable backbone assignment, transverse relaxation optimized spectroscopy (TROSY) [16][17][18] versions of HNCO [19], HNCA [19], HN(CO)CA [20], HNCACB [20] and HN(CO)CACB [20], using gradient echo-anti echo TROSY and 2 H-decoupling, were acquired together with a 2D 1 H- 15  Backbone assignment of MALT1 Casp-Ig3 (338-719) using the Targeted Acquisition (TA) approach A standard set of 3D TROSY-based triple resonance experiments with deuterium decoupling was acquired using iterative nonuniform sampling (NUS) [14], [22]. The TA procedure was used for automatical processing and analysis of spectra. TA allows a significant reduction in experiment and analysis time for sequential backbone assignment of proteins and has been described in detail previously [13][14][15]. Essentially, simultaneous co-processing with multidimensional decomposition (co-MDD) of all triple-resonance spectra [15] followed by the automated hyper-dimensional spectrum analysis generates clean peak lists with already prebuilt spin systems that can be efficiently used by automated assignment software AutoAssign [23]. The process is repeated (to accumulate more data in the experiments) and iterated until the assignment is sufficiently complete or converges. A 30% sampling schedule was used for the HNCO spectrum and a 19% sampling schedule was used for all other 3D spectra, yielding a total acquisition time of 136 hours (about 1 week). All spectra were acquired at 298 K on a Bruker Avance III HD 800 MHz spectrometer equipped with a 3mm CPTCI cryo-probe. Peak lists generated with the TA procedure were validated by manual analysis of the spectra in SPARKY [24]. Relevant parameters are displayed in Table 1.
Estimation of the secondary structure of MALT1 Casp-Ig3 (338-719) To estimate the secondary structure in MALT1 Casp-Ig3 (338-719), random coil chemical shifts corrected for nearest-neighbour effects [25] were subtracted from 13 C', 13 Cα and 13 Cβ chemical shifts corrected for deuterium isotope shifts [26] and plotted against the amino acid sequence of MALT1 Casp-Ig3 (338-719). The chemical shift index (CSI) for each carbon type was calculated using a cut-off at ± 0.7 and averaged over all three carbon types to a "consensus" CSI. The secondary structure estimated from the chemical shifts was then compared to the secondary structure of the previously reported X-ray structure of apo MALT1 Casp-Ig3 [8] (3V55) and of our in house X-ray structure of apo MALT1 Casp-Ig3 (data not published).

Evaluation of the TA approach
Apart from obtaining the backbone assignment of MALT1, the secondary goal of this project was to evaluate the performance of the TA approach for a large triple labelled protein. data points in individual backbone experiments. The peak counts do not level-off at the end of the TA procedure, suggesting that further measurements might improve the result of the assignment. However, the sample stability proved to be the limiting factor. Since the assignment was already obtained using the conventional approach, we decided that the TA data set accumulated during one week was sufficient for the purpose of evaluation of the TA procedure.
To make a fair comparison, it should be noted that the TA procedure in this work included modifications that could have benefited the conventional approach as well. These are running the experiment at higher magnetic field (800 MHz), which improves sensitivity and resolution, and use of an additional HN(CA)CO experiment that increases the success and reliability of assignment, albeit in expense of extending the total measurement time.
To evaluate the automated peak picking algorithm used in the TA procedure, all of the spectra were manually carefully analysed in SPARKY [24]. In Table 2 it is clearly seen that the TA approach was successful. Specifically, almost as many peaks were picked when using the TA approach as when using the conventional approach, using only one third of the acquisition time and only one NMR sample instead of two samples. Furthermore, during this measurement time, it was possible to include an additional experiment, the HN(CA)CO, which was essential to disambiguate assignments for several residues and correct a few errors in the assignment produced by the traditional approach.
It should be noted that peak picking in the HNCO spectrum is semi-automated and its correctness, which is insured by manual verification of the peaks in this spectrum, is crucial for the automated peak detection in all other spectra. Thus, the statistics on the quality of the peak lists refer to all spectra except for the HNCO. The number of false peaks in these spectra is lower than 0.8% in total. For an additional 0.6% of the peaks the obtained data does not allow discrimination between true or false. In addition to the automated peak list, careful manual peak picking allows finding approximately 5% to 20% more peaks and in case of the weakest spectrum, HN(CA)CO, almost 30% more peaks. This was expected, since the TA peak counts did not level-off in our data set (Fig 2).
Comparison of the backbone assignment obtained using the conventional and TA approaches is presented in Table 3. Overall the presented results clearly show that TA can be reliably used for in backbone assignment of large proteins. We clearly show that the method can be used to speed up data acquisition and analysis for backbone assignment of proteins with size of over 40 kDa.
The secondary structure of MALT1 Casp-Ig3 (338-719) The secondary chemical shifts of 13 C', 13 Cα and 13 Cβ were calculated and is shown in Fig 3  together with the "consensus" chemical shift index CSI. The CSI derived secondary structure was compared to the secondary structure of the apo MALT1 Casp-Ig3 crystal structures (unpublished in-house structure and published, PDB ID: 3V55), to validate the assignment. As can been seen in Fig 4 the secondary structure estimated from chemical shifts is close to identical to that of the X-ray structures. However some small differences are seen. Most of these differences, i.e. β1, gap in αC, gap in β3AB, gap between β2 Ã and β3 Ã can be explained by gaps in the assignment that creates gaps in the secondary structure estimation. These differences do not imply any real difference between the X-ray structure and the NMR assignment, merely that we do not have enough information to estimate the secondary structure from chemical shifts in these parts of the protein. Further β5 Ã was not identified as a β-strand based on the strict  criteria used for the "consensus" CSI. However, if the threshold is lowered slightly also this secondary structure element would be identified. All secondary structure elements except β1 and β5 Ig3 (denoted β5 Ã in Fig 4) could be assigned based on the data, i.e. αB, β2, αC, β3, β3A, β3B, αD, β4, loop 2 (L2), β5, loop 3 (L3), αE, αF, β6, α1 Ig3 , β1 Ig3 , β2 Ig3 , β3 Ig3 , β4 Ig3 , β6 Ig3 , β7 Ig3 . The different oligomerisation states of apo MALT1 Casp-Ig3 in solution (monomer) and crystals (dimer) are not reflected by great differences in secondary structure, suggesting that the secondary structure elements necessary for oligomerisation are pre-formed in solution.

Conclusions
In this work we present the NMR backbone assignment of apo MALT1 Casp-Ig3 in solution (monomer) and show that the secondary structure deduced from the chemical shifts resemble the secondary structure observed in the apo X-ray structures (dimer). In addition, we tested the performance of the TA assignment procedure for a large protein system and demonstrated for the first time clear benefit of the approach in reducing measurement and analysis time.
The nature of MALT1 activation and regulation suggests a highly dynamic system in solution. Key to understanding such system is to acquire knowledge on the conformational ensembles and their equilibrium to help elucidate the structure recognised by substrate. For ligand free MALT1 Casp-Ig3 this includes retrieving structure-dynamics information on the major flexible loops in the caspase domain, and their structural relationship with the C-terminal Ig3 domain. This necessitates solution-based techniques as a complement to X-ray crystallography. The 15 N/ 13 C/ 1 H backbone assignment of MALT1 Casp-Ig3 presented in this report forms a spring board to such future studies aimed at clarifying the role of MALT1 structuredynamics in substrate recognition and processing.