DYNLT (Tctex-1) forms a tripartite complex with dynein intermediate chain and RagA, hence linking this small GTPase to the dynein motor
FEBS JOURNAL (ISSN: 1742-464X) 282: (20) pp. 3945-3958. (2015)
It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a β strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.
The role of structural flexibility and stability in the interaction of serine proteases with their inhibitors
CURRENT PROTEIN AND PEPTIDE SCIENCE 16:(6) pp. 521-531. (2015)
Serine proteases and their natural inhibitors have long been served as excellent models for studying (primary, secondary and tertiary) structure - activity relationships of biologically interacting proteins. As protein flexibility has been accepted as a "fourth dimension" of the protein structure, its contribution to the binding process has gained much interest. In this article we review extreme cases of serine protease interactions with canonical serine protease inhibitors that provide unique insights into the dynamics of protein- protein interactions. The major conclusions of our review article are: a) taxon-specific inhibitory effects of two highly homologous protease inhibitors from Schistocerca gregaria (SGCI and SGTI), as investigated by H/D exchange experiments and NMR spectroscopy, are due to their differential flexibilities, b) stabilities of some protease and inhibitor complexes, the wide-spread and increased flexibility of some segments in the protein-protein complexes, as studied by X-ray crystallography and NMR-spectroscopy, appear to be proportional to the physical stability of the complex.
Different pH-sensitivity patterns of 30 sodium channel inhibitors suggest chemically different pools along the access pathway.
Front Pharmacol. 2015 Sep 25;6:210. doi: 10.3389/fphar.2015.00210. eCollection 2015.
The major drug binding site of sodium channels is inaccessible from the extracellular side, drug molecules can only access it either from the membrane phase, or from the intracellular aqueous phase. For this reason, ligand-membrane interactions are as important determinants of inhibitor properties, as ligand-protein interactions. One-way to probe this is to modify the pH of the extracellular fluid, which alters the ratio of charged vs. uncharged forms of some compounds, thereby changing their interaction with the membrane. In this electrophysiology study we used three different pH values: 6.0, 7.3, and 8.6 to test the significance of the protonation-deprotonation equilibrium in drug access and affinity. We investigated drugs of several different indications: carbamazepine, lamotrigine, phenytoin, lidocaine, bupivacaine, mexiletine, flecainide, ranolazine, riluzole, memantine, ritanserin, tolperisone, silperisone, ambroxol, haloperidol, chlorpromazine, clozapine, fluoxetine, sertraline, paroxetine, amitriptyline, imipramine, desipramine, maprotiline, nisoxetine, mianserin, mirtazapine, venlafaxine, nefazodone, and trazodone. We recorded the pH-dependence of potency, reversibility, as well as onset/offset kinetics. As expected, we observed a strong correlation between the acidic dissociation constant (pKa) of drugs and the pH-dependence of their potency. Unexpectedly, however, the pH-dependence of reversibility or kinetics showed diverse patterns, not simple correlation. Our data are best explained by a model where drug molecules can be trapped in at least two chemically different environments: A hydrophilic trap (which may be the aqueous cavity within the inner vestibule), which favors polar and less lipophilic compounds, and a lipophilic trap (which may be the membrane phase itself, and/or lipophilic binding sites on the channel). Rescue from the hydrophilic and lipophilic traps can be promoted by alkalic and acidic extracellular pH, respectively.
Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 112: pp. E3095-E3103. (2015)
Circular dichroism (CD) spectroscopy is a widely used technique for the study of protein structure. Numerous algorithms have been developed for the estimation of the secondary structure composition from the CD spectra. These methods often fail to provide acceptable results on α/β-mixed or β-structure-rich proteins. The problem arises from the spectral diversity of β-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual β-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the β-sheets account for the observed spectral diversity. We have developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures. This method can reliably distinguish parallel and antiparallel β-sheets and accurately estimates the secondary structure for a broad range of proteins. Moreover, the secondary structure components applied by the method are characteristic to the protein fold, and thus the fold can be predicted to the level of topology in the CATH classification from a single CD spectrum. By constructing a web server, we offer a general tool for a quick and reliable structure analysis using conventional CD or synchrotron radiation CD (SRCD) spectroscopy for the protein science research community. The method is especially useful when X-ray or NMR techniques fail. Using BeStSel on data collected by SRCD spectroscopy, we investigated the structure of amyloid fibrils of various disease-related proteins and peptides.
Evidence-based structural model of the Staphylococcal repressor protein: separation of functions into different domains
PLOS ONE 10:(9) Paper e0139086. 16 p. (2015)
Horizontal transfer of mobile genetic elements within Staphylococci is of high biomedical significance as such elements are frequently responsible for virulence and toxic effects. Staphylococcus-encoded repressor proteins regulate the replication of these mobile genetic elements that are located within the so-called pathogenicity islands. Here, we report structural and functional characterization of one such repressor protein, namely the Stl protein encoded by the pathogenicity island SaPIbov1. We create a 3D structural model and based on this prediction, we investigate the different functionalities of truncated and point mutant constructs. Results suggest that a helix-turn-helix motif governs the interaction of the Stl protein with its cognate DNA site: point mutations within this motif drastically decrease DNA-binding ability, whereas the interaction with the Stl-binding partner protein dUTPase is unperturbed by these point mutations. The 3D model also suggested the potential independent folding of a carboxy-terminal domain. This suggestion was fully verified by independent experiments revealing that the carboxy-terminal domain does not bind to DNA but is still capable of binding to and inhibiting dUTPase. A general model is proposed, which suggests that among the several structurally different repressor superfamilies Stl-like Staphylococcal repressor proteins belong to the helix-turn-helix transcription factor group and the HTH motif is suggested to reside within N-terminal segment.
Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1854:(10) pp. 1526-1535. (2015)
Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. An IgG1 molecule, which is now mainly used for antibody preparation, consists of a total of 12 immunoglobulin domains. Each domain has one disulfide bond. The CH3 domain is the C-terminal domain of the heavy chain of IgG1. The disulfide bonds of some of the CH3 domains are known to be reduced in recombinant human monoclonal antibodies. The lack of intramolecular disulfide bonds may decrease the stability and increase the aggregation propensity of an antibody molecule. To investigate the effects of a reduced disulfide bond in the CH3 domain on conformational stability and aggregation propensity, we performed several physicochemical measurements including circular dichroism, differential scanning calorimetry (DSC), and 2D NMR. DSC measurements showed that both the stability and reversibility of the reduced form were lower than those of the oxidized form. In addition, detailed analyses of the thermal denaturation data revealed that, although a dominant fraction of the reduced form retained a stable dimeric structure, some fractions assumed a less-specifically associated oligomeric state between monomers. The results of the present study revealed the characteristic aggregation properties of antibody molecules.
Supersaturation-limited and unlimited phase transitions compete to produce the pathway complexity in amyloid fibrillation.
JOURNAL OF BIOLOGICAL CHEMISTRY 290:(29) pp. 18134-18145. (2015)
Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.
A Multi-Pathway Perspective on Protein Aggregation: Implications for Control of the Rate and Extent of Amyloid Formation
FEBS LETTERS (ISSN: 0014-5793) (eISSN: 1873-3468) 589, DOI:10.1016/j.febslet.2015.01.032. (2015)
The nucleation-growth model has been used extensively for characterizing in vitro amyloid fibril formation kinetics and for simulating the relationship between amyloid and disease. In the majority of studies amyloid has been considered as the dominant, or sole, aggregation end product, with the presence of other competing non-amyloid aggregation processes, for example amorphous aggregate formation, being largely ignored. Here, we examine possible regulatory effects that off-pathway processes might exert on the rate and extent of amyloid formation - in particular their potential for providing false positives and negatives in the evaluation of anti-amyloidogenic agents. Furthermore, we investigate how such competing reactions might influence the standard interpretation of amyloid aggregation as a two-state system. We conclude by discussing our findings in terms of the general concepts of supersaturation and system metastability - providing some mechanistic insight as to how these empirical phenomena may manifest themselves in the amyloid arena.
Phosphorylation as Conformational Switch from the Native to Amyloid State - Trp-Cage as Protein Aggregation Model.
JOURNAL OF PHYSICAL CHEMISTRY B - CONDENSED MATTER MATERIALS SURFACES INTERFACES AND BIOPHYSICAL (ISSN: 1520-6106) (eISSN: 1520-5207) 119: DOI: 10.1021/jp5124234. (2015)
The 20 residue long Trp-cage miniprotein is an excellent model for both computational and experimental studies of protein folding and stability. Recently, great attention emerged to study disease-related protein misfolding, aggregation, and amyloid formation, with the aim of revealing their structural and thermodynamic background. Trp-cage is sensitive to both environmental and structure-modifying effects. It aggregates with ease upon structure destabilization, and thus it is suitable for modeling aggregation and amyloid formation. Here, we characterize the amyloid formation of several sequence modified and side-chain phosphorylated Trp-cage variants. We applied NMR, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, molecular dynamics (MD) simulations, and transmission electron microscopy (TEM) in conjunction with thioflavin-T (ThT) fluorescence measurements to reveal the structural consequences of side-chain phosphorylation. We demonstrate that the native fold is destabilized upon serine phosphorylation, and the resultant highly dynamic structures form amyloid-like ordered aggregates with high intermolecular β-structure content. The only exception is the D9S(P) variant, which follows an alternative aggregation process by forming thin fibrils, presenting a CD spectrum of PPII helix, and showing low ThT binding capability. We propose a complex aggregation model for these Trp-cage miniproteins. This model assumes an additional aggregated state, a collagen triple helical form that can precede amyloid formation. The phosphorylation of a single serine residue serves as a conformational switch, triggering aggregation, otherwise mediated by kinases in cell. We show that Trp-cage miniprotein is indeed a realistic model of larger globular systems of composite folding and aggregation landscapes and helps us to understand the fundamentals of deleterious protein aggregation and amyloid formation.