Familial Mutations and Post-translational Modifications of UCH-L1 in Parkinson's Disease and Neurodegenerative Disorders.

Parkinson's disease (PD) is one of the most common progressive neurodegenerative disorders in modern society. The disease involves many genetic risk factors as well as a sporadic pathogenesis that is age- and environment-dependent. Of particular interest is the formation of intra-neural fibrillar aggregates, namely Lewy bodies (LBs), the histological hallmark of PD, which results from aberrant protein homeostasis or misfolding that results in neurotoxicity. A better understanding of the molecular mechanism and composition of these cellular inclusions will help shed light on the progression of misfolding-associated neurodegenerative disorders. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is found to co-aggregate with α-synuclein (αS), the major component of LBs. Several familial mutations of UCH-L1, namely p.Ile93Met (p.I93M), p.Glu7Ala (p.E7A), and p.Ser18Tyr (p.S18Y), are associated with PD and other neurodegenerative disorders. Here, we review recent progress and recapitulate the impact of PD-associated mutations of UCH-L1 in the context of their biological functions gleaned from biochemical and biophysical studies. Finally, we summarize the effect of these genetic mutations and post-translational modifications on the association of UCHL1 and PD in terms of loss of cellular functions or gain of cellular toxicity.

A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation.

We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the α-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the ß-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the α-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases.

Towards multiparametric fluorescent imaging of amyloid formation: studies of a YFP model of alpha-synuclein aggregation.

Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimer's and Parkinson's diseases. In Parkinson's disease and several closely related syndromes, the protein alpha-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.

Evolution rescues folding of human immunodeficiency virus-1 envelope glycoprotein GP120 lacking a conserved disulfide bond.

The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel beta-strands in a six-stranded beta-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the beta-barrel fold, implying that beta-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two beta-strands that are important for the formation and/or integrity of the beta-barrel can be supported by either a disulfide bond or beta-sheet favoring residues.

The solution structure of the AppA BLUF domain: insight into the mechanism of light-induced signaling.

The transcriptional antirepressor AppA from the photosynthetic bacterium Rhodobacter sphaeroides senses both the light climate and the intracellular redox state. Under aerobic conditions in the dark, AppA binds to and thereby blocks the function of PpsR, a transcriptional repressor. Absorption of a blue photon dissociates AppA from PpsR and allows the latter to repress photosynthesis gene expression. The N terminus of AppA contains sequence homology to flavin-containing photoreceptors that belong to the BLUF family. Structural and chemical aspects of signal transduction mediated by AppA are still largely unknown. Here we present NMR studies of the N-terminal flavin-binding BLUF domain of AppA. Its solution structure adopts an alpha/beta-sandwich fold with a beta alpha beta beta alpha beta beta topology, which represents a new flavin-binding fold. Considerable disorder is observed for residues near the chromophore due to conformational exchange. This disorder is observed both in the dark and in the light-induced signaling state of AppA. Furthermore, we detect light-induced structural changes in a patch of surface residues that provide a structural link between light absorption and signal-transduction events.

Characterization and structural analyses of nonspecific lipid transfer protein 1 from mung bean.

Plant nonspecific lipid transfer proteins (nsLTPs) are thermal stable proteins that are capable of transferring lipid molecules between bilayers in vitro. This family of proteins, abundant in plants, is proposed to be involved in defense, pollination, and germination; the in vivo biological function remains, however, elusive. Here we report the purification and sequencing of an nsLTP1 from mung bean sprouts. We have also determined the solution structure of this nsLTP1, which represents the first 3D structure of the dicotyledonous nsLTP1 family. The global fold of mung bean nsLTP1 is similar to those of the monocotyledonous nsLTP1 structures and consists of four alpha-helices stabilized by four disulfide bonds. There are, however, some notable differences in the C-terminal tails and internal hydrophobic cavities. Circular dichroism and fluorescence spectroscopy were used to compare the thermodynamics and lipid transfer properties of mung bean nsLTP1 with those of rice nsLTP1. Docking of a lipid molecule into the solution structure of mung bean nsLTP1 reveals similar binding cavities and hydrophobic interactions as in rice nsLTP1, consistent with their comparable lipid transfer properties measured experimentally.

Entropy calculation of HIV-1 Env gp120, its receptor CD4, and their complex: an analysis of configurational entropy changes upon complexation.

The HIV-1 gp120/CD4 interaction shows a large, unprecedented entropy/enthalpy compensation, with the capacity to fine-tune recognition over a broad range of affinity. The intermolecular interaction involves stable hydrophobic contacts with a unique protruding CD4-Phe43 structure surrounded by an intermolecular hydrogen-bond network that covers the hemisphere of the CD4 D1 domain. We have applied a heuristic formula based on the covariance matrix of atom-positional fluctuations to assess the configurational entropy of the gp120/CD4 complex at different levels. The system was dissected into various subsets of atoms to evaluate the entropic contributions of different functional elements. By combining the trajectories of the free and complex forms, further insight into the conformational sampling was extracted. Despite the limited sampling time of 10 ns, the theoretically derived changes in configurational entropy are in fair agreement with the experimentally determined data. The simultaneous evaluation of different interaction modes through a decomposition approach is only feasible with the knowledge of the atomic trajectory of the system. The configurational entropy analysis in terms of combined trajectories presented here shall potentially provide accurate estimations of thermodynamic properties of biomolecules given sufficient sampling of conformational space.

The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics.

The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.

Atomic insight into the CD4 binding-induced conformational changes in HIV-1 gp120.

The entry of HIV-1 into a target cell requires gp120 and receptor CD4 as well as coreceptor CCR5/CXCR4 recognition events associated with conformational changes of the involved proteins. The binding of CD4 to gp120 is the initiation step of the whole process involving structural rearrangements that are crucial for subsequent pathways. Despite the wealth of knowledge about the gp120/CD4 interactions, details of the conformational changes occurring at this stage remain elusive. We have performed molecular dynamics simulations in explicit solvent based on the gp120/CD4/CD4i crystal structure in conjunction with modeled V3 and V4 loops to gain insight into the dynamics of the binding process. Three differentiated interaction modes between CD4 and gp120 were found, which involve electrostatics, hydrogen bond and van der Waals networks. A "binding funnel" model is proposed based on the dynamical nature of the binding interface together with a CD4-attraction gradient centered in gp120 at the CD4-Phe43-binding cavity. Distinct dynamical behaviors of free and CD4-bound gp120 were monitored, which likely represent the ground and pre-fusogenic states, respectively. The transition between these states revealed concerted motions in gp120 leading to: i) loop contractions around the CD4-Phe43-insertion cavity; ii) stabilization of the four-stranded "bridging sheet" structure; and iii) translocation and clustering of the V3 loop and the bridging sheet leading to the formation of the coreceptor binding site. Our results provide new insight into the dynamic of the underlying molecular recognition mechanism that complements the biochemical and structural studies.

NMR study of mersacidin and lipid II interaction in dodecylphosphocholine micelles. Conformational changes are a key to antimicrobial activity.

Mersacidin belongs to the type B lantibiotics (lanthionine-containing antibiotics) that contain post-translationally modified amino acids and cyclic ring structures. It targets the cell wall precursor lipid II and thereby inhibits cell wall synthesis. In light of the emerging antibiotics resistance problem, the understanding of the antibacterial activity on a structural basis provides a key to circumvent this issue. Here we present solution NMR studies of mersacidin-lipid II interaction in dodecylphosphocholine (DPC) micelles. Distinct solution structures of mersacidin were determined in three different states: in water/methanol solution and in DPC micelles with and without lipid II. The structures in various sample conditions reveal remarkable conformational changes in which the junction between Ala-12 and Abu-13 (where Abu is aminobutyric acid) effectively serves as the hinge for the opening and closure of the ring structures. The DPC micelle-bound form resembles the previously determined NMR and x-ray crystal structures of mersacidin in pure methanol but substantially deviates from the other two states in our current report. The structural changes delineate the large chemical shift perturbations observed during the course of a two-step (15)N-(1)H heteronuclear single quantum coherence titration. They also modulate the surface charge distribution of mersacidin suggesting that electrostatics play a central role in the mersacidin-lipid II interaction. The observed conformational adaptability of mersacidin might be a general feature of lipid II-interacting antibiotics/peptides.