Animal venom contains a mixture of toxic polypeptides that act in synergy to enable the fast immobilization of prey or deter predators and competitors. This polypeptide arsenal is constantly replenished by recruiting protein scaffolds from various organs into the venom gland. The recruited proteins often retain their ancestral functions but acquire specific modifications associated with novel toxic functions. Here we report an in-depth analysis of Hg1, a Kunitz-fold protease inhibitor with moderate blocking activity on Kv1.3, isolated from the venom gland of the Hadrurus gertschi scorpion. Electrophysiological analyses, supported by MD simulations, suggested that in contrast to canonical K+ blocking peptides, Hg1 does not insert a lysine residue into the channel pore. The crystal structure of the toxin revealed an unusually long, disordered extension at the C-terminal region of the molecule. Deletion of the C-terminal extension increased the potency of the toxin by order of magnitude and restored the voltage sensitivity of the block, a hallmark of the canonical block mechanism. In silico analysis supported the notion that removal of the disordered C-terminal extension has lifted a geometric restraint that prevents the wild-type toxin from effectively blocking Kv1.3. We propose that Hg1 is an evolutionary intermediate between a potent protease inhibitor and a potent K+ channel blocker.

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Å resolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi‐enzyme cellulosome complex, certain clostridia contain alternative sigma I (σ I ) factors that have cognate membrane‐associated anti‐σ I factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure‐function relationship of the extracellular sensory elements of Clostridium ( Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14‐superfamily motifs. The X‐ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI / rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σ I4 ‐dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σ I3 ‐dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation‐related genes. Structural similarity between clostridial PA14 dyads to PA14‐containing proteins in yeast helped identify another crucial signature element: the calcium‐binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14 A ) is dominant in directing the binding to the ligand in both bacteria. The two X‐ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.

Voltage gated K+ channels gate in response to changes in the electrical membrane potential by the coupling of a voltage sensing module with a K+-selective pore. During prolonged depolarizations, these channels enter a non-conductive slow-inactivated state that involves constriction of the channel pore. Recent studies suggest that the slow inactivated state is triggered by the penetration of water molecules into well-defined peripheral cavities surrounding the selectivity filter. Here we describe Cs1, a cone-snail toxin that blocks the drosophila shaker channel using a novel mode of action that shares common features with its inherent slow inactivation. Using double-mutant cycle analysis complemented by unconstrained docking and refinement by molecular-dynamics we obtained a docked toxin model, in which the toxin is bound off the pore axis. Analysis of the MD data suggested that Cs1 does not block the access of water molecules to the pore, but greatly modifies the access of water to the peripheral cavities. In concert with recent studies, we demonstrate that water permeation into these cavities is governed by a network of hydrogen-bonds formed at the extracellular face of the channel. The toxin specifically target these hydrogen bonds in three different channel subunits, by either eliminating or stabilizing them. The resulting imbalanced movement of water molecules around the selectivity filter triggers a series of molecular events culminating in an asymmetric collapse of the selectivity filter. A series of electrophysiological experiments utilizing D2O and various toxin/channel mutants that support the proposed mechanism will be described.

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.

Voltage gated ion channels gate in response to changes in the electrical membrane potential by the coupling of a voltage sensing module with an ion-selective pore. Toxins that target these channels are traditionally classified as either pore-blockers or gating-modifiers, the former bind and physically occlude the channel pore, while the later bind the voltage sensing module and restrict its movement in response to alterations in the membrane potential. Here we present Cs1, a kunitz-fold cone-snail toxin that blocks the Drosophila Shaker isoform with high affinity and seem to defy the traditional classification. We first obtained high resolution crystal structures of Cs1 and several of its mutants. Then, using site directed mutagenesis followed by double-mutant cycle analysis we identified key residue pairs in contact at the toxin-channel interface, which was clearly confined to the channel pore-module. Unconstrained rigid docking followed by a whole-atom molecular dynamics simulations yielded a model that was consistent with the experimental results, in which the toxin was bound off the pore axis and allowed the access of water molecules to the pore. Electrophysiological assays established that Cs1 does not dissociate from its binding site upon depolarization, and that its affinity for the channel is independent of the external K+ concentration, further setting it apart from the classical pore-blockers. Analysis of our MD simulations suggest that Cs1 blocks ion conductance using a novel allosteric mechanism that directly involves structural water molecules buried behind the selectivity filter of the channel.

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60-70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli . We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

Significance The human mitochondrial chaperonin is vital for proper cell function because it assists in folding of mitochondrial proteins. Additionally, it participates in extramitochondrial processes, such as apoptosis, inflammation, and carcinogenesis. In this study, we report the crystal structure of mitochondrial chaperonins. The model shows an “American football”-shaped intermediate, composed of two 7-membered chaperonin rings capped at each end by a cochaperonin ring. This complex was captured in the early stages of dissociation. The extensive interface contacts between its rings, the asymmetry that exists within each ring, and symmetric binding of nucleotide cofactors that exists between the rings suggest that the mitochondrial system operates via a mechanism that is distinct from the mechanism of the canonical Escherichia coli (GroEL/GroES) system.

The cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. Dockerin-bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin-borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface.

ABSTRACT The use of protein crystals as a source of nanoscale biotemplates has attracted growing interest in recent years owing to their inherent internal order. As these crystals are vulnerable to environmental changes, potential applications require their stabilization by chemical crosslinking. We have previously shown that such intermolecular chemical crosslinking reactions occurring within protein crystals are not random events, but start at preferred crosslinking sites imposed by the alignment of protein molecules and their packing within the crystalline lattice. Here we propose a new working hypothesis and demonstrate its feasibility in enabling us to extricate homogeneous populations of single protein molecules that display chemical point mutations or of dimers that show homogeneous chemical crosslinking, and that have the potential for isolation of higher structures. Characterization of the crosslinking mechanism and its end products opens the way to the potential retrieval of such specific modified/intermolecular crosslinked products simply by effecting partial crosslinking at identified preferred sites, followed by time‐controlled arrest of the crosslinking reaction and dissolution of the crystals by medium exchange complemented by chromatographic purification. Biotechnol. Bioeng. 2014;111: 1296–1303. © 2014 Wiley Periodicals, Inc.

The anaerobic, thermophilic, cellulosome-producing bacterium Clostridium thermocellum relies on a variety of carbohydrate-active enzymes in order to efficiently break down complex carbohydrates into utilizable simple sugars. The regulation mechanism of the cellulosomal genes was unknown until recently, when genomic analysis revealed a set of putative operons in C. thermocellum that encode σI factors (i.e. alternative σ factors that control specialized regulon activation) and their cognate anti-σI factor (RsgI). These putative anti-σI-factor proteins have modules that are believed to be carbohydrate sensors. Three of these modules were crystallized and their three-dimensional structures were solved. The structures show a high overall degree of sequence and structural similarity to the cellulosomal family 3 carbohydrate-binding modules (CBM3s). The structures of the three carbohydrate sensors (RsgI-CBM3s) and a reference CBM3 are compared in the context of the structural determinants for the specificity of cellulose and complex carbohydrate binding. Fine structural variations among the RsgI-CBM3s appear to result in alternative substrate preferences for each of the sensors.

Ruminococcus flavefaciens is a cellulolytic bacterium found in the rumen of herbivores and produces one of the most elaborate and variable cellulosome systems. The structure of an R. flavefaciens protein (RfCohG, ZP_06142108), representing a freestanding (non-cellulosomal) type III cohesin module, has been determined. A selenomethionine derivative with a C-terminal histidine tag was crystallized and diffraction data were measured to 2.44 Å resolution. Its structure was determined by single-wavelength anomalous dispersion, revealing eight molecules in the asymmetric unit. RfCohG exhibits the most complex among all known cohesin structures, possessing four α-helical elements and a topographical protuberance on the putative dockerin-binding surface.

The mitochondrial Hsp60-Hsp10 complex assists the folding of various proteins impelled by ATP hydrolysis, similar to the bacterial chaperonins GroEL and GroES. The near-atomic structural details of the mitochondrial chaperonins are not known, despite the fact that almost two decades have passed since the structures of the bacterial chaperonins became available. Here, the crystallization procedure, diffraction experiments and structure determination by molecular replacement of the mammalian mitochondrial chaperonin HSP60 (E321K mutant) and its co-chaperonin Hsp10 are reported.

Determination in the past and the contemporary re-determination of several protein structures are considered in the context of our personal experience with the use of single-wavelength anomalous dispersion (SAD) techniques. A unifying feature of these cases is that in each event, the anomalous signal generated by metal ions was not optimized to the peak of the absorption edge, but was, nevertheless sufficient for structure re-determination. Examples are described with the emphasis on educational aspects. Determinants of success are discussed in each case. In addition, briefly discussed are some concepts related to the quality of the diffraction data used in experimental phasing, with special attention to SAD phasing.

The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD-CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs.