Surface-exposed Hydrophobic Residues Research Articles

MipZ caps the plus-end of FtsZ polymers to promote their rapid disassembly

The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.

Blocking PSD95-PDZ3's amyloidogenesis through point mutations that inhibit high-temperature reversible oligomerization (RO).

The third PDZ domain of the postsynaptic density protein 95 (PSD95-PDZ3; 11 kDa, 103 residues) has a propensity to form amyloid fibrils at high temperatures. At neutral pH, PDZ3 is natively folded, but it exhibits a peculiar three-state thermal unfolding with a reversible oligomerization (RO) equilibrium at high temperatures, which is uncharacteristic in the unfolding of a small globular protein as PDZ3 is. Here, we examined the RO's role in PDZ3's amyloidogenesis at high-temperature using two variants (F340A and L342A) that suppress the high-temperature RO and five single-alanine-mutated variants, where we mutated surface-exposed hydrophobic residues to alanine. Circular Dichroism (CD), Analytical Ultracentrifuge (AUC), and other spectroscopic measurements confirmed the retention of the native structure at ambient temperature. Differential Scanning Calorimetry (DSC) was used to assess the presence or absence of the high-temperature RO, and the amyloidogenicity of the variants was measured by Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM). By comparing the fraction of RO and the ThT signal, we found that mutations that suppressed the high-temperature RO strongly inhibited amyloidogenesis. On the other hand, all variants forming RO also formed amyloids under the same conditions as the wild-type PDZ3.

Disrupting putative N-glycosylation site N17 in lipase Lip11 of Yarrowia lipolytica yielded a catalytically efficient and thermostable variant accompanying conformational changes

Lip11 gene from oleaginous yeast Yarrowia lipolytica MSR80 was recombinantly expressed in Pichia pastoris X33. Native secretion signal present in its sequence resulted in 92 % expression in comparison to α-secretion factor which resulted to 900 U/L in the extracellular broth. Catalytic triad in Lip11, like most lipases, was formed by serine, histidine, and aspartate residues. While point mutation disrupting putative glycosylation site (N389) present towards the C-terminus ruinously effected its stability and catalytic activity, disruption of the first putative glycosylation site (N17) located towards the N-terminus presented interesting insights. Mutation resulted in a variant N1 exhibiting higher thermal and acid stability; a t1/2 of 198 min was obtained at 50 °C and it retained almost 80 % activity following incubation at pH 3. Catalytic efficiency was improved by 2.7 fold and a 10 °C rise in temperature optima was accompanied by higher relative activity in acidic range. Thermal stability corresponded to convoying structural modifications in the tertiary structure, findings of fluorescence spectroscopy suggested. Thermal fluorescence studies revealed a Tm of 65 °C for both Lip11 and N1 and λmax of Lip11 exhibited a blue shift upon refolding while no shift in the λmax of N1 was observed. A resilient tertiary structure which could fold back to its native confirmation upon thermal denaturation and increase in surface-exposed hydrophobic residues as revealed by ANS binding assay summed up to thermal stability of N1. Furthermore, circular dichroism data disclosed an alternate ratio of alpha-helices and beta-sheets; respective values changed from 36 % and 8%–27% and 19 %. Following mutation, substrate specificity remained unaffected and similar to native protein, N1 showed activation in presence of organic solvents and most divalent cations.

Identifying weak interdomain interactions that stabilize the supertertiary structure of the N-terminal tandem PDZ domains of PSD-95

Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface-exposed hydrophobic residues. Both interfaces were confirmed experimentally. Proximity of interdomain contacts to the binding pockets may explain the observed coupling between conformation and binding. The low-energy barrier between conformations allows submillisecond dynamics, which were time-averaged in previous NMR and FRET studies. Moreover, the small contact interfaces were likely overridden by lattice contacts as crystal structures were rarely sampled in simulations. Our hybrid approach can identify transient interdomain interactions, which are abundant in multidomain proteins yet often obscured by dynamic averaging.

Polyethylene glycol promotes autoxidation of cytochrome c

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.

Membrane binding of the insertion sequence of Proteus vulgaris L-amino acid deaminase stabilizes protein structure and increases catalytic activity

Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity.

Conserved hydrophobic residues in the CARP/β-sheet domain of cyclase-associated protein are involved in actin monomer regulation.

Cyclase-associated protein (CAP) is a multidomain protein that promotes actin filament dynamics. The C-terminal region of CAP contains a CAP and X-linked retinitis pigmentosa 2 protein (CARP) domain (or a β-sheet domain), which binds to actin monomer and is essential for enhancing exchange of actin-bound nucleotides. However, how the CARP domain binds to actin is not clearly understood. Here, we report that conserved hydrophobic residues in the CARP domain play important roles in the function of CAP to regulate actin dynamics. Single mutations of three conserved surface-exposed hydrophobic residues in the CARP domain of CAS-2, a Caenorhabditis elegans CAP, significantly reduce its binding to actin monomers and suppress its nucleotide exchange activity on actin. As a result, these mutants are weaker than wild-type to compete with ADF/cofilin to promote recycling of actin monomers for polymerization. A double mutation (V367A/I373A) eliminates these actin-regulatory functions of CAS-2. These hydrophobic residues and previously identified functional residues are scattered on a concave β-sheet of the CARP domain, suggesting that a wide area of the β-sheet is involved in binding to actin. These observations suggest that the CARP domain of CAP binds to actin in a distinct manner from other known actin-binding proteins.

Aggregation as a consequence of glycation: insight into the pathogenesis of arthritis.

Advanced glycation end products (AGEs) as a result of Maillard reaction are currently at the heart of the pathogenesis of several diseases and hence are the objective of numerous investigations. Glycation of proteins has been an implication in long-term complications. Collagen is the most abundant protein in the human body. The purpose of this study was to monitor and characterize the oligomeric aggregates and AGEs of human collagen on addition of glyoxal using ultraviolet, fluorescence, circular dichroism (CD) spectroscopy, docking studies, ITC, and microscopy. Collagen was incubated for varying time periods up to 21days with three different concentrations (5, 20, and 40mM) of glyoxal. Collagen exists as molten globule at day 6, evident from native-like secondary structure, altered tryptophan, and high ANS fluorescence due to surface-exposed hydrophobic residues. Glycated collagen as AGEs and aggregates was observed at day 18 and 21, respectively. Formation of AGE and aggregates were confirmed by UV and fluorescence spectroscopy. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine) forming the Schiff base, the carboxymethyl lysine, and carbonyl content. Non-tryptophan fluorescence for AGEs was also monitored as the emission peak at 400 and 440nm, respectively. SEM and TEM confirmed the oligomeric nature of aggregates. Glyoxal at 40mM shows maximum alterations in protein structure followed by 20 and 5mM concentration. In the present paper, we propose that a high concentration of glyoxal for a prolonged time results in the formation of harmful aggregates and AGEs.

Thermal Induction of an Alternatively Folded State in Human IgG-Fc

We report the formation of a non-native, folded state of human IgG4-Fc induced by a high temperature at neutral pH and at a physiological salt concentration. This structure is similar to the molten globule state in that it displays a high degree of secondary structure content and surface-exposed hydrophobic residues. However, it is highly resistant to chemical denaturation. The thermally induced state of human IgG4-Fc is thus associated with typical properties of the so-called alternatively folded state previously described for murine IgG, IgG-Fab, and individual antibody domains (V(L), V(H), C(H)1, and C(H)3) under acidic conditions in the presence of anions. Like some of these molecules, human IgG4-Fc in its alternative fold exists as a mixture of different oligomeric structures, dominated by an equilibrium between monomeric and heptameric species. Heating further induces the formation of fibrous structures in the micrometer range.

Hsp90 Is Required for Transfer of the Cholera Toxin A1 Subunit from the Endoplasmic Reticulum to the Cytosol

Cholera toxin (CT) is an AB(5) toxin that moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). The driving force for CTA1 dislocation into the cytosol is unknown. Here, we demonstrate that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor. CT activity against cultured cells and ileal loops was also blocked by GA, as was the ER-to-cytosol export of CTA1. Experiments using RNA interference or N-ethylcarboxamidoadenosine, a drug that inhibits ER-localized GRP94 but not cytosolic Hsp90, confirmed that the inhibitory effects of GA resulted specifically from the loss of Hsp90 activity. This work establishes a functional role for Hsp90 in the ERAD-mediated dislocation of CTA1.

Surface-Exposed Hydrophobic Residues on Small Ankyrin-1 Mediate Binding to Obscurin

Small ankyrin-1 (sAnk1, Ank1.5) is a splice variant of the ANK1 gene that binds to the large modular protein, obscurin A, with nanomolar affinity, a reaction that may help to organize the sarcoplasmic reticulum in striated muscle. A subset of lysine and arginine residues in the 2 ankyrin repeats of sAnk1 interact specifically with 4 glutamate residues in a stretch of 30 amino acids of obscurin to mediate binding. Homology modeling and molecular dynamics simulations have revealed a “hot spot” of 4 hydrophobic residues exposed on the surface of the ankyrin repeat domain of sAnk1. We used site-directed mutagenesis of bacterially expressed fusion proteins, followed by blot overlays and surface plasmon resonance assays, to study the contribution of these 4 residues, V70, F71, I102 and I103, to binding to the 30-mer of obscurin. Alanine mutations of each of these four residues inhibited binding to residues 6316-6345 of obscurin (Obsc6316-6345). In contrast, V70A and I102A mutations had no effect on binding to a second sAnk1 binding site on obscurin, located within residues 6231-6260 (Obsc6231-6260 ). Using the same methods, we mutated the 5 hydrophobic residues present in Obsc6316-6345 to alanine and identified V6328, I6332, and V6334 as critical for proper binding. Our results suggest that hydrophobic interactions as well as electrostatic interactions are important for the binding of sAnk1 to Obsc6316-6345, consistent with studies of the complexes formed by other ankyrin repeat proteins with their ligands. Hydrophobic interactions are likely to contribute to the difference in affinity of sAnk1 for Obsc6316-6345 and Obsc6231-6260, and for the dominant role played by the more C-terminal sequence in binding. Supported by grant R01- AR056330 from the NIH to RJB and training grants T32 GM08181 (to RJB) and T32 AR07592 (to M. Schneider).

Distinct Acidic Clusters and Hydrophobic Residues in the Alternative Splice Domains X1 and X2 of α7 Integrins Define Specificity for Laminin Isoforms

The binding specificity of α7β1 integrins for different laminin isoforms is defined by the X1 and X2 splice domains located in the beta-propeller domain of the α7 subunit. In order to gain insight into the mechanism of specific laminin–integrin interactions, we defined laminin-binding epitopes of the α7X1 and -X2 domains by single amino acid substitutions and domain swapping between X1 and X2. The interaction of mutated, recombinantly prepared α7X1β1 and α7X2β1 heterodimers with various laminin isoforms was studied by surface plasmon resonance and solid phase binding assays. The data show that distinct clusters of surface-exposed acidic residues located in different positions of the X1 and the X2 loops are responsible for the specific recognition of laminins. These residues are conserved between the respective X1 or X2 splice domains of the α7 chains of different species, some also in the corresponding X1/X2 splice domains of α6 integrin. Interestingly, ligand binding was also modulated by mutating surface-exposed hydrophobic residues (α7X1L205, α7X2Y208) at positions corresponding to the fibronectin binding synergy site in α5β1 integrin. Mutations in X1 that affected binding to laminin-1 also affected binding to laminin-8 and -10, but not to the same extent, thus allowing conclusions on the specific role of individual surface epitopes in the selective recognition of laminin-1 versus laminins -8 and -10. The role of the identified epitopes was confirmed by molecular dynamics simulations of wild-type integrins and several inactivating mutations. The analysis of laminin isoform interactions with various X1/X2 chimaera lend further support to the key role of negative surface charges and pointed to an essential contribution of the N-terminal TARVEL sequence of the X1 domain for recognition of laminin-8 and -10. In conclusion, specific surface epitopes containing charged and hydrophobic residues are essential for ligand binding and define specific interactions with laminin isoforms.

An attempt to define allergen-specific molecular surface features: a bioinformatic approach

Allergens are proteins that elicit T helper lymphocyte type 2 (Th2) responses culminating in IgE antibody production and allergic disease. However, we have no answer to the fundamental question of why certain proteins are allergens, while others are not. We hypothesized that analysis of the surface of diverse allergens may reveal common structural features which might enable them to be recognized as Th2-inducing antigens by cells of the innate immune system. We have therefore used the ConSurf server to search for allergen-specific motifs. This has enabled us to identify residue conservation patterns in the homologues of Ara t 8 (plant profilin), Act c 1 (actinidin), Bet v 1 (plant pathogenesis-related protein) and Ves v 5 (venom allergen). The results demonstrate the presence of allergen-specific patches consisting of an unusually high proportion of surface-exposed hydrophobic residues. The patches that have been identified may represent molecular patterns recognizable by cells of the innate immune system.

Structure-based substitutions for increased solubility of a designed protein.

Manipulation of protein solubility is important for many aspects of protein design and engineering. Previously, we designed a series of consensus ankyrin repeat proteins containing one, two, three and four identical repeats (1ANK, 2ANK, 3ANK and 4ANK). These proteins, particularly 4ANK, are intended for use as a universal scaffold on which specific binding sites can be constructed. Despite being well folded and extremely stable, 4ANK is soluble only under acidic conditions. Designing interactions with naturally occurring proteins requires the designed protein to be soluble at physiological pH. Substitution of six leucines with arginine on exposed hydrophobic patches on the surface of 4ANK resulted in increased solubility over a large pH range. Study of the pH dependence of stability demonstrated that 4ANK is one of the most stable ankyrin repeat proteins known. In addition, analogous leucine to arginine substitutions on the surface of 2ANK allowed the partially folded protein to assume a fully folded conformation. Our studies indicate that replacement of surface-exposed hydrophobic residues with positively charged residues can significantly improve protein solubility at physiological pH.

Crystal Structure of a C-terminal Fragment of Growth Arrest-specific Protein Gas6: RECEPTOR TYROSINE KINASE ACTIVATION BY LAMININ G-LIKE DOMAINS

Receptor tyrosine kinases of the Axl family are activated by Gas6, the product of growth arrest-specific gene 6. Gas6-Axl signaling is implicated in cell survival, adhesion, and migration. The receptor-binding site of Gas6 is located within a C-terminal pair of laminin G-like (LG) domains that do not resemble any other receptor tyrosine kinase ligand. We report the crystal structure at 2.2-A resolution of a Gas6 fragment spanning both LG domains (Gas6-LG). The structure reveals a V-shaped arrangement of LG domains strengthened by an interdomain calcium-binding site. LG2 of Gas6-LG contains two unusual features: an alpha-helix cradled by one edge of the LG beta-sandwich and a conspicuous patch of surface-exposed hydrophobic residues. Mutagenesis of some residues in this patch reduces Gas6-LG binding to the extracellular domain of Axl as well as Axl activation in glioblastoma cells, identifying a component of the receptor-binding site of Gas6.

Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1.

Kalata B1 is a member of a new family of polypeptides, isolated from plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However, in the synthesis involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution. In addition to facilitating folding to a compact stable structure, cyclization has an important effect on biological activity as assessed by hemolytic activity.

Insights into the Molecular Basis of Salt Tolerance from the Study of Glutamate Dehydrogenase from Halobacterium salinarum

A homology-based modeling study on the extremely halophilic glutamate dehydrogenase from Halobacterium salinarum has been used to provide insights into the molecular basis of salt tolerance. The modeling reveals two significant differences in the characteristics of the surface of the halophilic enzyme that may contribute to its stability in high salt. The first of these is that the surface is decorated with acidic residues, a feature previously seen in structures of halophilic enzymes. The second is that the surface displays a significant reduction in exposed hydrophobic character. The latter arises not from a loss of surface-exposed hydrophobic residues, as has previously been proposed, but from a reduction in surface-exposed lysine residues. This is the first report of such an observation.