Physicochemical Properties and Route of Systemic Delivery Control the In Vivo Dynamics and Breakdown of Radiolabeled Gold Nanostars.

The in vivo dynamics of nanoparticles requires a mechanistic understanding of multiple factors. Here, for the first time, the surprising breakdown of functionalized gold nanostars (F-AuNSs) conjugated with antibodies and 64 Cu radiolabels in vivo and in artificial lysosomal fluid ex vivo, is shown. The short-term biodistribution of F-AuNSs is driven by the route of systemic delivery (intravenous vs intraperitoneal) and long-term fate is controlled by the tissue type in vivo. In vitro studies including endocytosis pathways, intracellular trafficking, and opsonization, are combined with in vivo studies integrating a milieu of spectroscopy and microcopy techniques that show F-AuNSs dynamics is driven by their physicochemical properties and route of delivery. F-AuNSs break down into sub-20 nm broken nanoparticles as early as 7 days postinjection. Martini coarse-grained simulations are performed to support the in vivo findings. Simulations suggest that shape, size, and charge of the broken nanoparticles, and composition of the lipid membrane depicting various tissues govern the interaction of the nanoparticles with the membrane, and the rate of translocation across the membrane to ultimately enable tissue clearance. The fundamental study addresses critical gaps in the knowledge regarding the fate of nanoparticles in vivo that remain a bottleneck in their clinical translation.

Resolving the conformational dynamics of ErbB growth factor receptor dimers.

The combinatorial dimerization of the ErbB growth factor receptors (ErbB1- ErbB4) are critical for their function. Here, we have characterized the conformational dynamics of ErbB transmembrane homo-dimers and hetero-dimers by using a coarse-grain simulation framework. All dimers, except ErbB4-4 and ErbB1-4, exhibit at least two conformations. The reported NMR structures correspond to one of these conformations, representing the N-terminal active state in ErbB1-1 (RH2), ErbB2-2 (RH1) and ErbB4-4 (RH) homo-dimers and the LH dimer in ErbB3-3 homo-dimer, validating the computational approach. Further, we predict a right-handed ErbB3-3 dimer conformer that warrants experimental testing. The five hetero-dimers that have not yet been experimentally resolved display prominent right-handed dimers associating by the SmXXXSm motif. Our results provide insights into the constitutive signaling of ErbB4 after cleavage of the extracellular region. The presence of the inactive-like dimer conformers leading to symmetric kinase domains gives clues on the autoinhibition of the receptor dimers. The dimer states characterized here represent an important step towards understanding the combinatorial cross associations in the ErbB family.

Structural dynamics of Smoothened (SMO) in the ciliary membrane and its interaction with membrane lipids.

The Smoothened receptor (SMO, a 7 pass transmembrane domain, Class F GPCR family protein) plays a crucial role in the Hedgehog (HH) signaling pathway, which is involved in embryonic development and is implicated in various types of cancer throughout the animal kingdom. In the absence of HH signaling, SMO is inhibited by Patched 1 (PTC1; a 12 pass transmembrane domain protein), which is localized in the primary cilia. HH binding leads to the dislocation of PTC1 from the cilia, thus making way for SMO to localize in the primary cilia, as an essential prerequisite for its activation. We have carried out MARTINI coarse-grained molecular dynamics simulations of SMO in POPC and in ciliary membrane models, respectively, to study the interactions of SMO with cholesterol and other lipid molecules in the ciliary membrane, and to gain molecular-level insights into the role of the primary cilia in shaping the functional dynamics of SMO. We are able to identify the interaction of membrane cholesterols with definite sites and domains within SMO and relate them with known cholesterol-binding sequence and structure motifs. We show that cholesterol interactions with the transmembrane domain TMD, unlike those with the cysteine-rich domain (CRD) and the intracellular domain (ICD), are through residues belonging to known cholesterol-binding motifs. Notably, a few persistent interactions of cholesterol with lower TM cholesterol-binding domains are governed by the presence of multiple cholesterol-binding motifs. These analyses have also helped to identify and define a strict cholesterol consensus motif (CCM), which may well steer cholesterol into the hitherto identified binding sites within the TMD of SMO. We have also reported the interaction of phosphatidylinositol 4-phosphate with the intracellular region of transmembrane (TM) helices (TM1, TM3, TM4, and TM5), intracellular loop1, helix8, and Arg/Lys clusters of the ICD. Structural analysis of SMO domains shows significant changes in the CRD and ICD, during the course of the simulation. Further detailed analysis of the dynamics of the TMD reveals the movements of TM5, TM6, and TM7, linked with the helix8, which are possibly involved in shaping the conformational disposition of the ICD. The movement of these TM helices could possibly be a consequence of interactions involving the extracellular domain and extracellular loops. In addition, our analysis also shows that phosphatidylinositol-4-phosphate (PI4P), along with some ICD cholesterols, are implicated in anchoring SMO in the membrane.

Lipid-dependent conformational landscape of the ErbB2 growth factor receptor dimers.

Altered lipid metabolism has been linked to cancer development and progression. Several roles have been attributed to the increased saturation and length of lipid acyl tails observed in tumors, but its effect on signaling receptors is still emerging. In this work, we have analyzed the lipid dependence of the ErbB2 growth factor receptor dimerization that plays an important role in the pathogenesis of breast cancer. We have performed coarse-grain ensemble molecular dynamics simulations to comprehensively sample the ErbB2 monomer-dimer association. Our results indicate a dynamic dimer state with a complex conformational landscape that is modulated with increasing lipid tail length. We resolve the native N-terminal "active" and C-terminal "inactive" conformations in all membrane compositions. However, the relative population of the N-terminal and C-terminal conformers is dependent on length of the saturated lipid tails. In short-tail membranes, additional non-specific dimers are observed which are reduced or absent in long-tailed bilayers. Our results indicate that the relative population as well as the structure of the dimer state is modulated by membrane composition. We have correlated these differences to local perturbations of the membrane around the receptor. Our work is an important step in characterizing ErbB dimers in healthy and diseased states and emphasize the importance of sampling lipid dynamics in understanding receptor association.