The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD’s inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.
Structural biology is a field that enables a better understanding of proteins from scratch. From the available techniques, solution NMR is one well established that provides structure, dynamics and protein-molecules interaction. In a NMR lab routine, from data acquisition until protein/mechanisms elucidation comes a process that can undergo months. During the past decades, different tools were developed for NMR data processing, peaks assignment, structure elucidation and data submission. Since many of these programs demand great computational skills, a few groups have tried to combine those programs and make them more friendly and useful, what can possibilite a faster process. Here we highlight CCPNMR2.4 analysis and ARIA2.3, responsible for peak assignment and structure calculation, respectively, and can work associated. Although being academic free and the possibility of working with a GUI interface, the common N-terminal acetylation and C-terminal amidation modifications are not implemented in a way that possibilities to work with them in combination, what results in a dilemma. This work brings visual data that evidences the low usability of CCPN and ARIA with N-terminal acetylated and C-terminal amidated proteins and propose a workflow to overcome this problem, which may improve the usage of both software in the mentioned versions and facilitate the lab users already used to these programs. As a proof of concept, we have chosen a N-terminal amidated peptide, L-Phenylseptin, whose structure has already been solved with other programs. Statistical data shows that no significant difference was found with the structure obtained with the new protocol. In conclusion, we exhibit a new protocol that can be used in combination with CCPNMR2.4 and ARIA2.3 for protein with the mentioned modifications and it successfully works and manipulates these molecules.
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