Effect of the Macromolecular Crowding Agents on the Structure and Function of Human Arginase-I, a Therapeutically Important Enzyme.

Macromolecular crowding has been known to influence the structure and function of many enzymes through excluded volume effects and/or soft interactions. Here, we employed two synthetic macromolecular crowders, Dextrans and poly(ethylene glycol)s (PEGs) with varying molecular masses, to examine how they affected the structure and function of a therapeutically important enzyme, human arginase-I that catalyzes the conversion of l-arginine to l-ornithine and urea. Except at greater concentrations of Dextran 200, Dextrans were observed to slightly reduce the enzymatic activity, indicating that they exert their influence mainly through the excluded volume effects. Similar outcomes were seen with PEGs, with the exception of PEG 1000, where the activity decreased with increasing PEG concentrations, showing the maximum effect at a 20 g/L concentration. This finding suggests that the enzyme function is reduced by the soft interactions of this macromolecule with the enzyme, supported by the binding measurement. Secondary and local tertiary structures and thermodynamic stability were also affected, suggesting that PEG 1000 has an impact on the protein's structure. Furthermore, molecular dynamics simulation studies suggest that the catalytic pocket is disturbed, presumably by the unwinding of neighboring helix 9. As a result, the positioning of nearby Glu277 is altered, which prevents His141 and Glu277 from making contact. This hampers the proton transfer from the catalytic His141 to the intermediate species to form ornithine, a crucial step for the substrate hydrolysis reaction by this arginase. Overall, the knowledge gained from this study might be helpful for understanding how different enzymes work in a crowded/cellular environment.

Metal ions-induced stability and function of bimetallic human arginase-I, a therapeutically important enzyme.

Recent studies have highlighted the therapeutic importance of bimetallic human arginase-I against hyperargininemia and L-arginine auxotrophic cancers. The longer retention of catalytic activity of the Co2+-enzyme than that of the Mn2+ in human serum is associated with its enhanced therapeutic potential. To understand the basis of this and also to explore the role of a bimetallic center as well as the role of individual metal ions in the stability, we performed a detailed biochemical and biophysical investigation. The thermodynamic and kinetic stabilities of both the holo proteins are found to be significantly higher than the apo form, indicating that an intact bimetallic centre is vital for the enhanced stability of the holo proteins. The Co2+-protein is found to be more stable than that of the Mn2+, which might explain its longer retention of activity observed in the serum. Mutational studies demonstrated that the metal ions are individually crucial for both the enhanced stability and catalytic activity. Furthermore, we investigated the underlying mechanism for the effect of heat activation on the holo protein for higher catalytic activity, which is not yet known for arginases. Our data reveal that heat activation significantly increases the stability of the holo protein through a metal-induced increase in the helical content leading to the formation of a kinetically competent enzyme. Thus, the present study provides an in-depth insight into the significance of heat activation and the role of metal ions in human arginase, which may be useful for better understanding of its therapeutic use.

Unravelling the binding mechanism and protein stability of human serum albumin while interacting with nefopam analogues: a biophysical and insilico approach.

In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in the blood. Its binding affinity and concomitant changes in its conformation, binding site and simulations were also studied. Fluorescence data revealed that the fluorescence quenching of HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs binding constants (KA) are in the order of NF3 > NF2 > NF1 with values of 1.53 ± .057 × 104, 2.16 ± .071 × 104 and 3.6 ± .102 × 105 M-1, respectively. Concurrently, thermodynamic parameters indicate that the binding process was spontaneous, and the complexes were stabilized mostly by hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with hydrophobic interactions. Circular dichroism (CD) studies revealed that there is a decrease in α-helix with an increase in ß-sheets and random coils signifying partial unfolding of the protein upon binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular docking studies showed that NF1, NF2 and NF3 bound to subdomains IIIA, IB and IIA through hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with LYS 413. Furthermore, molecular simulations unveiled that NFs binding was in support with the structural perturbation observed in CD, which is evident from the root mean square deviation and Rg fluctuations. We hope our insights will provide ample scope for engineering new drugs based on the resemblances with NFs for enhanced efficacy with HSA.

New players in the same old game: a system level in silico study to predict type III secretion system and effector proteins in bacterial genomes reveals common themes in T3SS mediated pathogenesis.

BACKGROUND: Type III secretion system (T3SS) plays an important role in virulence or symbiosis of many pathogenic or symbiotic bacteria [CHM 2:291-294, 2007; Physiology (Bethesda) 20:326-339, 2005]. T3SS acts like a tunnel between a bacterium and its host through which the bacterium injects 'effector' proteins into the latter [Nature 444:567-573, 2006; COSB 18:258-266, 2008]. The effectors spatially and temporally modify the host signalling pathways [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe5:571-579, 2009]. In spite its crucial role in host-pathogen interaction, the study of T3SS and the associated effectors has been limited to a few bacteria [Cell Microbiol 13:1858-1869, 2011; Nat Rev Microbiol 6:11-16, 2008; Mol Microbiol 80:1420-1438, 2011]. Before one set out to perform systematic experimental studies on an unknown set of bacteria it would be beneficial to identify the potential candidates by developing an in silico screening algorithm. A system level study would also be advantageous over traditional laboratory methods to extract an overriding theme for host-pathogen interaction, if any, from the vast resources of data generated by sequencing multiple bacterial genomes. RESULTS: We have developed an in silico protocol in which the most conserved set of T3SS proteins was used as the query against the entire bacterial database with increasingly stringent search parameters. It enabled us to identify several uncharacterized T3SS positive bacteria. We adopted a similar strategy to predict the presence of the already known effectors in the newly identified T3SS positive bacteria. The huge resources of biochemical data [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe 5:571-579, 2009; BMC Bioinformatics 7(11):S4, 2010] on the T3SS effectors enabled us to search for the common theme in T3SS mediated pathogenesis. We identified few cellular signalling networks in the host, which are manipulated by most of the T3SS containing pathogens. We went on to look for correlation, if any, between the biological quirks of a particular class of bacteria with the effectors they harbour. We could pin point few effectors, which were enriched in certain classes of bacteria. CONCLUSION: The current study would open up new avenues to explore many uncharacterized T3SS positive bacteria. The experimental validation of the predictions from this study will unravel a generalized mechanism for T3SS positive bacterial infection into host cell.