Insight derived from molecular dynamics simulation into substrate-induced changes in protein motions of proteinase K.

Because of the significant industrial, agricultural and biotechnological importance of serine protease proteinase K, it has been extensively investigated using experimental approaches such as X-ray crystallography, site-directed mutagenesis and kinetic measurement. However, detailed aspects of enzymatic mechanism such as substrate binding, release and relevant regulation remain unstudied. Molecular dynamics (MD) simulations of the proteinase K alone and in complex with the peptide substrate AAPA were performed to investigate the effect of substrate binding on the dynamics/molecular motions of proteinase K. The results indicate that during simulations the substrate-complexed proteinase K adopt a more compact and stable conformation than the substrate-free form. Further essential dynamics (ED) analysis reveals that the major internal motions are confined within a subspace of very small dimension. Upon substrate binding, the overall flexibility of the protease is reduced; and the noticeable displacements are observed not only in substrate-binding regions but also in regions opposite the substrate-binding groove/pockets. The dynamic pockets caused by the large concerted motions are proposed to be linked to the substrate recognition, binding, orientation and product release; and the significant displacements in regions opposite the binding groove/pockets are considered to play a role in modulating the dynamics of enzyme-substrate interaction. Our simulation results complement the biochemical and structural studies, highlighting the dynamic mechanism of the functional properties of proteinase K.

Expression, purification and crystal structure of a truncated acylpeptide hydrolase from Aeropyrum pernix K1.

Acylpeptide hydrolase (APH) catalyzes the N-terminal hydrolysis of Nalpha-acylpeptides to release Nalpha-acylated amino acids. The crystal structure of recombinant APH from the thermophilic archaeon Aeropyrum pernix K1 (apAPH) was reported recently to be at a resolution of 2.1 Angstrom; using X-ray diffraction. A truncated mutant of apAPH that lacks the first short alpha-helix at the N-terminal, apAPH-delta(1-21), was cloned, expressed, characterized and crystallized. Data from biochemical experiments indicate that the optimum temperature of apAPH is decreased by 15 degrees C with the deletion of the N-terminal alpha-helix. However, the enzyme activity at the optimal temperature does not change. It suggests that this N-terminal alpha-helix is essential for thermostability. Here, the crystal structure of apAPH-delta(1-21) has been determined by molecular replacement to 2.5 Angstrom;. A comparison between the two structures suggests a difference in thermostability, and it can be concluded that by adding or deleting a linking structure (located over different domains), the stability or even the activity of an enzyme can be modified.

[Using GST-tag to capture protein interaction of an interferon-inducible systemic lupus associated gene, IFIT1].

OBJECTIVE: To investigate the protein-to-protein interaction of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), a newly discovered systemic lupus erythematosus (SLE) related up-regulated gene, and its possible function. METHODS: Peripheral blood of 40 SLE patients was obtained to extract total RNA and synthesized cDNA. Real-time PCR was used to determine the IFIT1 expression at transcript level. Peripheral blood of another 10 SLE patients was extracted to obtain specimens of white blood cell lysate. Molecular cloning and a modified gluthion S-transferase (GST)-pull down assay were used to capture the protein interacting with IFIT1 in the specimens of white blood cell lysate. MALDI-TOF mass spectrometry (MS) was preformed to identify the captured protein that could interact with IFIT1. Twenty-nine sex and age-matched healthy persons were used as controls. RESULTS: By real-time PCR showed that the IFIT1Delta Ct value (x +/- s) was 2.344 +/- 1.200 in the SLE patients and was 3.734 +/- 1.274 in the controls (P < 0.001), showing a significant up-regulation in SLE patients. IFIT1 was cloned and GST-IFIT1 fusion protein was expressed in Escherichia coli. GST-IFIT1 fusion protein was further purified using Glutathione Sepharose 4B column, and was treated as bait to capture prey from peripheral white blood cell lysate of SLE patients. MALDI-TOF MS detected protein interaction between Rho/Rac guanine nucleotide exchange factor and IFIT1. CONCLUSION: IFIT1 may interact with Rho/Rac guanine nucleotide exchange factor, and regulate the activation of Rho/Rac proteins, thus being involved in the pathogenesis of SLE.