From Discrete to Continuum Models of Three-Dimensional Deformations in Epithelial Sheets

Metamorphic proteins, including proteins with high levels of sequence identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% sequence identity adopt the same fold. Which topologies can be bridged by these highly identical sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high sequence identity (80 and 77%, respectively) with engineered variants of protein G (G_A, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial sequence modification. Furthermore, the MBP variant (80% identical to G_A) remained active. Thermodynamic analysis of numerous G_A and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.     

Smooth Functional Transition along a Mutational Pathway with an Abrupt Protein Fold Switch

Recent protein design experiments have demonstrated that proteins can migrate between folds through the accumulation of substitution mutations without visiting disordered or nonfunctional points in sequence space. To explore the biophysical mechanism underlying such transitions we use a three-letter continuous protein model with seven atoms per amino acid to provide realistic sequence-structure and sequence-function mappings through explicit simulation of the folding and interaction of model sequences. We start from two 16-amino-acid sequences folding into an α-helix and a β-hairpin, respectively, each of which has a preferred binding partner with 35 amino acids. We identify a mutational pathway between the two folds, which features a sharp fold switch. By contrast, we find that the transition in function is smooth. Moreover, the switch in preferred binding partner does not coincide with the fold switch. Discovery of new folds in evolution might therefore be facilitated by following fitness slopes in sequence space underpinned by binding-induced conformational switching.     

Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function,Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.     

Genomic and Structural Characterization of Kunitz-Type Peptide LmKTT-1a Highlights Diversity and Evolution of Scorpion Potassium Channel Toxins

Background

Recently, a new subfamily of long-chain toxins with a Kunitz-type fold was found in scorpion venom glands. Functionally, these toxins inhibit protease activity and block potassium channels. However, the genomic organization and three-dimensional (3-D) structure of this kind of scorpion toxin has not been reported.

Principal Findings

Here, we characterized the genomic organization and 3-D nuclear magnetic resonance structure of the scorpion Kunitz-type toxin, LmKTT-1a, which has a unique cysteine pattern. The LmKTT-1a gene contained three exons, which were interrupted by two introns located in the mature peptide region. Despite little similarity to other Kunitz-type toxins and a unique pattern of disulfide bridges, LmKTT-1a possessed a conserved Kunitz-type structural fold with one α-helix and two β-sheets. Comparison of the genomic organization, 3-D structure, and functional data of known toxins from the α-KTx, β-KTx, γ-KTx, and κ-KTx subfamily suggested that scorpion Kunitz-type potassium channel toxins might have evolved from a new ancestor that is completely different from the common ancestor of scorpion toxins with a CSα/β fold. Thus, these analyses provide evidence of a new scorpion potassium channel toxin subfamily, which we have named δ-KTx.

Conclusions/Significance

Our results highlight the genomic, structural, and evolutionary diversity of scorpion potassium channel toxins. These findings may accelerate the design and development of diagnostic and therapeutic peptide agents for human potassium channelopathies.     

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)₄ complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)₂ lobes joined with D₂ symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)₄ complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)₄ kinase core, because a β₄ subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.     

How a Spatial Arrangement of Secondary Structure Elements Is Dispersed in the Universe of Protein Folds

It has been known that topologically different proteins of the same class sometimes share the same spatial arrangement of secondary structure elements (SSEs). However, the frequency by which topologically different structures share the same spatial arrangement of SSEs is unclear. It is important to estimate this frequency because it provides both a deeper understanding of the geometry of protein folds and a valuable suggestion for predicting protein structures with novel folds. Here we clarified the frequency with which protein folds share the same SSE packing arrangement with other folds, the types of spatial arrangement of SSEs that are frequently observed across different folds, and the diversity of protein folds that share the same spatial arrangement of SSEs with a given fold, using a protein structure alignment program MICAN, which we have been developing. By performing comprehensive structural comparison of SCOP fold representatives, we found that approximately 80% of protein folds share the same spatial arrangement of SSEs with other folds. We also observed that many protein pairs that share the same spatial arrangement of SSEs belong to the different classes, often with an opposing N- to C-terminal direction of the polypeptide chain. The most frequently observed spatial arrangement of SSEs was the 2-layer α/β packing arrangement and it was dispersed among as many as 27% of SCOP fold representatives. These results suggest that the same spatial arrangements of SSEs are adopted by a wide variety of different folds and that the spatial arrangement of SSEs is highly robust against the N- to C-terminal direction of the polypeptide chain.     

Structure Based Annotation of Helicobacter pylori Strain 26695 Proteome

The availability of complete genome sequences of H. pylori 26695 has provided a wealth of information enabling us to carry out in silico studies to identify new molecular targets for pharmaceutical treatment. In order to construe the structural and functional information of complete proteome, use of computational methods are more relevant since these methods are reliable and provide a solution to the time consuming and expensive experimental methods. Out of 1590 predicted protein coding genes in H. pylori, experimentally determined structures are available for only 145 proteins in the PDB. In the absence of experimental structures, computational studies on the three dimensional (3D) structural organization would help in deciphering the protein fold, structure and active site. Functional annotation of each protein was carried out based on structural fold and binding site based ligand association. Most of these proteins are uncharacterized in this proteome and through our annotation pipeline we were able to annotate most of them. We could assign structural folds to 464 uncharacterized proteins from an initial list of 557 sequences. Of the 1195 known structural folds present in the SCOP database, 411 (34% of all known folds) are observed in the whole H. pylori 26695 proteome, with greater inclination for domains belonging to α/β class (36.63%). Top folds include P-loop containing nucleoside triphosphate hydrolases (22.6%), TIM barrel (16.7%), transmembrane helix hairpin (16.05%), alpha-alpha superhelix (11.1%) and S-adenosyl-L-methionine-dependent methyltransferases (10.7%).     

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

Almost all proteins that reside in the outer membrane (OM) of Gram-negative bacteria contain a membrane-spanning segment that folds into a unique β barrel structure and inserts into the membrane by an unknown mechanism. To obtain further insight into outer membrane protein (OMP) biogenesis, we revisited the surprising observation reported over 20 years ago that the Escherichia coli OmpA β barrel can be assembled into a native structure in vivo when it is expressed as two noncovalently linked fragments. Here, we show that disulfide bonds between β strand 4 in the N-terminal fragment and β strand 5 in the C-terminal fragment can form in the periplasmic space and greatly increase the efficiency of assembly of “split” OmpA, but only if the cysteine residues are engineered in perfect register (i.e., they are aligned in the fully folded β barrel). In contrast, we observed only weak disulfide bonding between β strand 1 in the N-terminal fragment and β strand 8 in the C-terminal fragment that would form a closed or circularly permutated β barrel. Our results not only demonstrate that β barrels begin to fold into a β-sheet-like structure before they are integrated into the OM but also help to discriminate among the different models of OMP biogenesis that have been proposed.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Sequential Unfolding of Beta Helical Protein by Single-Molecule Atomic Force Microscopy

The parallel βhelix is a common fold among extracellular proteins, however its mechanical properties remain unexplored. In Gram-negative bacteria, extracellular proteins of diverse functions of the large ‘TpsA’ family all fold into long βhelices. Here, single-molecule atomic force microscopy and steered molecular dynamics simulations were combined to investigate the mechanical properties of a prototypic TpsA protein, FHA, the major adhesin of Bordetella pertussis. Strong extension forces were required to fully unfold this highly repetitive protein, and unfolding occurred along a stepwise, hierarchical process. Our analyses showed that the extremities of the βhelix unfold early, while central regions of the helix are more resistant to mechanical unfolding. In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified. This nucleus harbors structural elements packed against the βhelix that might contribute to stabilizing the N-terminal region of FHA. Hierarchical unfolding of the βhelix in response to a mechanical stress may maintain β-helical portions that can serve as templates for regaining the native structure after stress. The mechanical properties uncovered here might apply to many proteins with β-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.     

Altered Backbone and Side-Chain Interactions Result in Route Heterogeneity during the Folding of Interleukin-1β (IL-1β)

Deletion of the β-bulge trigger-loop results in both a switch in the preferred folding route, from the functional loop packing folding route to barrel closure, as well as conversion of the agonist activity of IL-1β into antagonist activity. Conversely, circular permutations of IL-1β conserve the functional folding route as well as the agonist activity. These two extremes in the folding-functional interplay beg the question of whether mutations in IL-1β would result in changes in the populations of heterogeneous folding routes and the signaling activity. A series of topologically equivalent water-mediated β-strand bridging interactions within the pseudosymmetric β-trefoil fold of IL-1β highlight the backbone water interactions that stabilize the secondary and tertiary structure of the protein. Additionally, conserved aromatic residues lining the central cavity appear to be essential for both stability and folding. Here, we probe these protein backbone-water molecule and side chain-side chain interactions and the role they play in the folding mechanism of this geometrically stressed molecule. We used folding simulations with structure-based models, as well as a series of folding kinetic experiments to examine the effects of the F42W core mutation on the folding landscape of IL-1β. This mutation alters water-mediated backbone interactions essential for maintaining the trefoil fold. Our results clearly indicate that this perturbation in the primary structure alters a structural water interaction and consequently modulates the population of folding routes accessed during folding and signaling activity.     

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

α-Galactosidases catalyze the hydrolysis of terminal α-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae α-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 Å resolution. The monomer folds into a catalytic (α/β)₈ barrel and a C-terminal β-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the β-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.     

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from Bdellovibrio bacteriovorus–that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.     

Slow and Bimolecular Folding of a De Novo Designed Monomeric Protein DS119

De novo protein design offers a unique means to test and advance our understanding of how proteins fold. However, most current design methods are native structure eccentric and folding kinetics has rarely been considered in the design process. Here, we show that a de novo designed mini-protein DS119, which folds into a βαβ structure, exhibits unusually slow and concentration-dependent folding kinetics. For example, the folding time for 50 μM of DS119 was estimated to be ∼2 s. Stopped-flow fluorescence resonance energy transfer experiments further suggested that its folding was likely facilitated by a transient dimerization process. Taken together, these results highlight the need for consideration of the entire folding energy landscape in de novo protein design and provide evidence suggesting nonnative interactions can play a key role in protein folding.     

Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the α/β-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the α/β-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the α/β-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.     

Mechanical Resistance in Unstructured Proteins

Single-molecule pulling experiments on unstructured proteins linked to neurodegenerative diseases have measured rupture forces comparable to those for stable folded proteins. To investigate the structural mechanisms of this unexpected force resistance, we perform pulling simulations of the amyloid β-peptide (Aβ) and α-synuclein (αS), starting from simulated conformational ensembles for the free monomers. For both proteins, the simulations yield a set of rupture events that agree well with the experimental data. By analyzing the conformations occurring shortly before rupture in each event, we find that the mechanically resistant structures share a common architecture, with similarities to the folds adopted by Aβ and αS in amyloid fibrils. The disease-linked Arctic mutation of Aβ is found to increase the occurrence of highly force-resistant structures. Our study suggests that the high rupture forces observed in Aβ and αS pulling experiments are caused by structures that might have a key role in amyloid formation.     

Alternative Splice Variants in TIM Barrel Proteins from Human Genome Correlate with the Structural and Evolutionary Modularity of this Versatile Protein Fold

After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)₈ barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)₂, (βα)₄, (βα)₅, (βα)₆, and (βα)₇ subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the proteins encoded in the human genome.     

Interdomain contacts control folding of transcription factor RfaH

Escherichia coli RfaH activates gene expression by tethering the elongating RNA polymerase to the ribosome. This bridging action requires a complete refolding of the RfaH C-terminal domain (CTD) from an α-helical hairpin, which binds to the N-terminal domain (NTD) in the free protein, to a β-barrel, which interacts with the ribosomal protein S10 following RfaH recruitment to its target operons. The CTD forms a β-barrel when expressed alone or proteolytically separated from the NTD, indicating that the α-helical state is trapped by the NTD, perhaps co-translationally. Alternatively, the interdomain contacts may be sufficient to drive the formation of the α-helical form. Here, we use functional and NMR analyses to show that the denatured RfaH refolds into the native state and that RfaH in which the order of the domains is reversed is fully functional in vitro and in vivo. Our results indicate that all information necessary to determine its fold is encoded within RfaH itself, whereas accessory factors or sequential folding of NTD and CTD during translation are dispensable. These findings suggest that universally conserved RfaH homologs may change folds to accommodate diverse interaction partners and that context-dependent protein refolding may be widespread in nature.