Luminescent and paramagnetic properties of nanoparticles shed light on their interactions with proteins

Nanoparticles have been recognized as promising tools for targeted drug-delivery and protein therapeutics. However, the mechanisms of protein-nanoparticle interaction and the dynamics underlying the binding process are poorly understood. Here, we present a general methodology for the characterization of protein-nanoparticle interaction on a molecular level. To this end we combined biophysical techniques including nuclear magnetic resonance (NMR), circular dichroism (CD), resonance energy transfer (RET) and surface plasmon resonance (SPR). Particularly, we analyzed molecular mechanisms and dynamics of the interaction of CaF2nanoparticles with the prototypical calcium sensor calmodulin (CaM). We observed the transient formation of an intermediate encounter complex involving the structural region linking the two domains. Specific interaction of CaM with CaF2NPs is driven by the N-terminal EF-hands, which seem to recognize Ca2+on the surface of the nanoparticle. We conclude that CaF2NP-CaM interaction is fully compatible with potential applications in nanomedicine. Overall, the methods presented in this work can be extended to other systems and may be useful to quantitatively characterize structural and dynamic features of protein-NP interactions with important implications for nanomedicine and nano-biotechnology

A novel Porphyromonas gingivalis enzyme: An atypical dipeptidyl peptidase III with an ARM repeat domain

Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins

Hypoxia Inducible Lipid Droplet Associated protein inhibits Adipose Triglyceride Lipase.

Elaborate control mechanisms of intracellular triacylglycerol (TAG) breakdown are critically involved in the maintenance of energy homeostasis. Hypoxia inducible lipid droplet associated protein (HILPDA)/Hypoxia inducible gene-2 (Hig-2) has been shown to affect intracellular TAG levels, yet, the underlying molecular mechanisms are unclear. Here, we show that HILPDA inhibits adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of intracellular TAG hydrolysis. HILPDA shares structural similarity with G0/G1 switch gene 2 (G0S2), an established inhibitor of ATGL. HILPDA inhibits ATGL activity in a dose-dependent manner with an IC50 value of 2 M. ATGL inhibition depends on the direct physical interaction of both proteins and involves the N- terminal, hydrophobic region of HILPDA and the N-terminal, patatin-domain containing segment of ATGL. Finally, confocal microscopy combined with Foerster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) analysis indicated that HILPDA and ATGL colocalize and physically interact intracellularly. These findings provide a rational biochemical explanation for the tissue-specific increased TAG accumulation in HILPDA overexpressing transgenic mouse models

Crystal structure of the Saccharomyces cerevisiae monoglyceride lipase Yju3p

AbstractMonoglyceride lipases (MGLs) are a group of α/β-hydrolases that catalyze the hydrolysis of monoglycerides (MGs) into free fatty acids and glycerol. This reaction serves different physiological functions, namely in the last step of phospholipid and triglyceride degradation, in mammalian endocannabinoid and arachidonic acid metabolism, and in detoxification processes in microbes. Previous crystal structures of MGLs from humans and bacteria revealed conformational plasticity in the cap region of this protein and gave insight into substrate binding. In this study, we present the structure of a MGL from Saccharomyces cerevisiae called Yju3p in its free form and in complex with a covalently bound substrate analog mimicking the tetrahedral intermediate of MG hydrolysis. These structures reveal a high conservation of the overall shape of the MGL cap region and also provide evidence for conformational changes in the cap of Yju3p. The complex structure reveals that, despite the high structural similarity, Yju3p seems to have an additional opening to the substrate binding pocket at a different position compared to human and bacterial MGL. Substrate specificities towards MGs with saturated and unsaturated alkyl chains of different lengths were tested and revealed highest activity towards MG containing a C18:1 fatty acid

A) SDS page analysis of purified recombinant <i>Pg</i>DPP III wt (1–886 aa, left and the N-terminal DPP III fragment (1–679 aa, right; B) Far-UV spectra of the full length protein and the DPP III fragment.

<p>A) SDS page analysis of purified recombinant <i>Pg</i>DPP III wt (1–886 aa, left and the N-terminal DPP III fragment (1–679 aa, right; B) Far-UV spectra of the full length protein and the DPP III fragment.</p

Overlay of the ligand free structures of <i>Pg</i>DPP III generated during MD simulations.

<p>Overlay of the initial structure of PgDPP III (obtained by comparative modelling, redand the structure obtained after 200 ns of MD simulations (blue). The zinc ion is represented as a grey sphere.</p

Overlay of Arg₂-2NA and Arg₂-AMC bound in the active site of <i>Pg</i>DPP III iThe final structures obtained by simulation of the <i>Pg</i>DPP III—Diarginyl arylamide substrates complexes are aligned.

<p>Substrates are shown as sticks representation and the protein structures as cartoon representations, colored yellow (<i>Pg</i>DPP III—Arg₂-2NA, simulated 200 ns) and gray (<i>Pg</i>DPP III—Arg₂-AMC, simulated 150 ns), respectively. The Zn²⁺ ion is represented as a gray sphere.</p

Orientation of Arg₂-2NA in the active site of <i>Pg</i>DPP III determined by separate simulations of two replicas of the <i>Pg</i>DPP III-Arg₂-2NA complex.

<p>The replica obtained at the end of 200 ns of MD simulation is colored yellow and replica obtained after 150 ns of MD simulations violet). The Zn²⁺ ion is represented as a gray sphere.</p

Isothermal titration calorimetry measurements of the E433A variant of <i>Pg</i>DPP III with A) angiotensin II, B) tynorphin and C) IVYPW.

<p>The upper panels show the time course evolution of heat for each injection and the bottom panels show peak integration as a function of molar ratio of E433A <i>Pg</i>DPP III (inactive variant) to all three ligands. The data were corrected by subtraction of an appropriate blank experiment and fit with nonlinear regression. The solid curves represent the best fits using a one binding site model.</p