The structure of the protein known both as neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), and also as the 8 kDa dynein light chain (LC8) has been solved by X-ray diffraction. Two PIN/LC8 monomers related by a two-fold axis form a rectangular dimer. Two pairs of alpha-helices cover opposite faces, and each pair of helices packs against a beta-sheet with five antiparallel beta-strands. Each five-stranded beta-sheet contains four strands from one monomer and a fifth strand from the other monomer. A 13-residue peptide from nNOS is bound to the dimer in a deep hydrophobic groove as a sixth antiparallel beta-strand. The structure provides key insights into dimerization of and peptide binding by the multifunctional PIN/LC8 protein.
"ZDOCK generated 2000 conformations of DYNLL1-Pilin complex and the top 500 models of complex were visualized in JMOL . Interestingly, almost all models exhibited a similar pattern of Pilin interaction with the reported DYNLL1 binding pocket , , . Desolvation energy (−9.40 kcal/mol) and a global free energy (−20.76 kcal/mol) (Figure 3) profile indicated a high interaction probability of DYNLL1 and Pilin. "
[Show abstract][Hide abstract] ABSTRACT: Cytoplasmic dynein light chain 1 (DYNLL1) is a component of large protein complex, which is implicated in cargo transport processes, and is known to interact with many cellular and viral proteins through its short consensus motif (K/R)XTQT. Still, it remains to be explored that bacterial proteins also exhibit similar recognition sequences to make them vulnerable to host defense mechanism. We employed multiple docking protocols including AUTODOCK, PatchDock, ZDOCK, DOCK/PIERR and CLUSPRO to explore the DYNLL1 and Pilin interaction followed by molecular dynamics simulation assays. Subsequent structural comparison of the predicted binding site for DYNLL1-Pilin complex against the experimentally verified DYNLL1 binding partners was performed to cross check the residual contributions and to determine the binding mode. On the basis of in silico analysis, here we describe a novel interaction of DYNLL1 and receptor binding domain of Pilin (the main protein constituent of bacterial type IV Pili) of gram negative bacteria Pseudomonas aeruginosa (PAO), which is the third most common nosocomial pathogen associated with the life-threatening infections. Evidently, our results underscore that Pilin specific motif (KSTQD) exhibits a close structural similarity to that of Vaccinia virus polymerase, P protein Rabies and P protein Mokola viruses. We speculate that binding of DYNLL1 to Pilin may trigger an uncontrolled inflammatory response of the host immune system during P. aeruginosa chronic infections thereby opening a new pioneering area to investigate the role of DYNLL1 in gram negative bacterial infections other than viral infections. Moreover, by manifesting a strict correspondence between sequence and function, our study anticipates a novel drug target site to control the complications caused by P. aeruginosa infections.
PLoS ONE 09/2013; 8(10):e76730. DOI:10.1371/journal.pone.0076730 · 3.23 Impact Factor
"DLC8, one of the three dimeric light chains of the dynein complex, is a highly conserved protein with diverse functions ,. It acts as an adaptor between dynein and its cargo proteins , but may also have motor-independent functions in activities such as protein folding and dimerization, protein-protein interactions, and nuclear import of proteins , , , . We have found that a large region of CDK5RAP2 encompassing the DLC8-binding domain is required for association with dynein. "
[Show abstract][Hide abstract] ABSTRACT: CDK5RAP2 is a centrosomal protein known to be involved in the regulation of the γ-tubulin ring complex and thus the organization of microtubule arrays. However, the mechanism by which CDK5RAP2 is itself recruited to centrosomes is poorly understood. We report here that CDK5RAP2 displays highly dynamic attachment to centrosomes in a microtubule-dependent manner. CDK5RAP2 associates with the retrograde transporter dynein-dynactin and contains a sequence motif that binds to dynein light chain 8. Significantly, disruption of cellular dynein-dynactin function reduces the centrosomal level of CDK5RAP2. These results reveal a key role of the dynein-dynactin complex in the dynamic recruitment of CDK5RAP2 to centrosomes.
PLoS ONE 07/2013; 8(7):e68523. DOI:10.1371/journal.pone.0068523 · 3.23 Impact Factor
"Based on the observation that all dynein subunits and the dynactin p150Glued subunit are predicted to be homodimeric , we hypothesized that the IC and p150Glued fragments interact through a local symmetry axis, forming a hetero-tetrameric complex, or a ‘dimer of dimers’, which implies that the regions comprising the interface of one or both components must be monomeric in the absence of the other preceding the interaction. This hypothesis stems from ‘symmetry’ considerations and is supported by multiple crystal structures of ‘dimer of dimers’ interactions (e.g., transcription factors bound to palindromic DNA , the IC peptide binding to LC8 , EB1 binding to the CAP-gly domain of p150Glued
, etc.). Specifically, in forming a homodimeric coiled coil, two binding surfaces are created (see Fig. 5A). "
[Show abstract][Hide abstract] ABSTRACT: Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.
PLoS ONE 04/2013; 8(4):e59453. DOI:10.1371/journal.pone.0059453 · 3.23 Impact Factor
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