Plasmid replication initiator RepB forms a hexamer reminiscent of ring helicases and has mobile nuclease domains

RepB initiates plasmid rolling‐circle replication by binding to a triple 11‐bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. The structure of native full‐length RepB reveals a hexameric ring molecule, where each protomer has two domains. The origin‐binding and catalytic domains show a three‐layer α–β–α sandwich fold. The active site is positioned at one of the faces of the β‐sheet and coordinates a Mn²⁺ ion at short distance from the essential nucleophilic Y99. The oligomerization domains (ODs), each consisting of four α‐helices, together define a compact ring with a central channel, a feature found in ring helicases. The toroidal arrangement of RepB suggests that, similar to ring helicases, it encircles one of the DNA strands during replication to confer processivity to the replisome complex. The catalytic domains appear to be highly mobile with respect to ODs. This mobility may account for the adaptation of the protein to two distinct DNA recognition sites.     

PackHelix: a tool for helix-sheet packing during protein structure prediction

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Predicting the topology and constructing the geometry of structural motifs involving α‐helices and/or β‐strands are therefore key steps for accurate prediction of protein structure. While many efforts have focused on how to pack helices and on how to sample exhaustively the topologies and geometries of multiple strands forming a β‐sheet in a protein, there has been little progress on generating native‐like packings of helices on sheets. We describe a method that can generate the packing of multiple helices on a given β‐sheet for αβα sandwich type protein folds. This method mines the results of a statistical analysis of the conformations of αβ₂ motifs in protein structures to provide input values for the geometric attributes of the packing of a helix on a sheet. It then proceeds with a geometric builder that generates multiple arrangements of the helices on the sheet of interest by sampling through these values and performing consistency checks that guarantee proper loop geometry between the helices and the strands, minimal number of collisions between the helices, and proper formation of a hydrophobic core. The method is implemented as a module of ProteinShop. Our results show that it produces structures that are within 4–6 Å RMSD of the native one, regardless of the number of helices that need to be packed, though this number may increase if the protein has several helices between two consecutive strands in the sequence that pack on the sheet formed by these two strands. Proteins 2011; Published 2011 Wiley‐Liss, Inc.     

Determination of the secondary structural elements of chicken liver fatty acid binding protein by two‐dimensional homonuclear NMR

A conformational study in solution of the fatty acid binding protein from chicken liver is presented. The nearly complete sequence‐specific ¹H resonance assignment was achieved from homonuclear two‐dimensional nmr experiments using a sample of native protein. The principal elements of secondary structure were identified: 10 antiparallel β‐strands and one helical segment followed by a turn comprising 5 residues. These elements correspond closely with those of the crystal structure of the related protein, and two new secondary structural features obtained from the nmr data are the β‐sheet conformation between the first and the last β‐strand in the protein sequence, as well as a helical loop at the N‐terminus of the polypeptide chain. © 1999 John Wiley & Sons, Inc. Biopoly 50: 1–11, 1999     

Optimization of a β‐sheet‐cap for long loop closure

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: R W ITVTI – loop – KKIRV W E). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: R W ITVTI‐(GGGGKK)₂GGGG‐KKIRV W E) remaining folded (ΔG_U = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: R W ITVRI W ‐(GGGGKK)₂GGGG‐ W KTIRV W E) supports a remarkably stable β‐sheet (ΔG_U = 6.3 kJ/mol at 295 K, T_m = ～55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.     

BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

The ensemble folding kinetics of the FBP28 WW domain revealed by an all-atom Monte Carlo simulation in a knowledge-based potential

In this work, we apply a detailed all‐atom model with a transferable knowledge‐based potential to study the folding kinetics of Formin‐Binding protein, FBP28, which is a canonical three‐stranded β‐sheet WW domain. Replica exchange Monte Carlo simulations starting from random coils find native‐like (Cα RMSD of 2.68 Å) lowest energy structure. We also study the folding kinetics of FBP28 WW domain by performing a large number of ab initio Monte Carlo folding simulations. Using these trajectories, we examine the order of formation of two β‐hairpins, the folding mechanism of each individual β‐hairpin, and transition state ensemble (TSE) of FBP28 WW domain and compare our results with experimental data and previous computational studies. To obtain detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Further, a rigorous P_fold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated Φ values. Our analysis shows that the turn structure between first and second β strands is a partially stable structural motif that gets formed before entering the TSE in FBP28 WW domain and there exist two major pathways for the folding of FBP28 WW domain, which differ in the order and mechanism of hairpin formation. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

NMR structure of rALF‐Pm3, an anti‐lipopolysaccharide factor from shrimp: Model of the possible lipid A‐binding site

The anti‐lipopolysaccharide factor ALF‐Pm3 is a 98‐residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter as a folded and ¹⁵N uniformly labeled rALF‐Pm3 protein. Its 3D structure was established by NMR and consists of three α‐helices packed against a four‐stranded β‐sheet. The C³⁴? C⁵⁵ disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF‐Pm3 binds to LPS, lipid A and to OM®‐174, a soluble analogue of lipid A. Biophysical studies of rALF‐Pm3/LPS and rALF‐Pm3/OM®‐174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF‐Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A‐binding site of ALF‐Pm3. Such a binding site, located on the ALF‐Pm3 β‐sheet and involving seven charged residues, is well conserved in ALF‐L from Limulus polyphemus and in ALF‐T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that β‐hairpin synthetic peptides corresponding to ALF‐L β‐sheet bind to LPS. Delineating lipid A‐binding site of ALFs will help go further in the de novo design of new antibacterial or LPS‐neutralizing drugs. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 207–220, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

An amino acid code for β‐sheet packing structure

To understand the relationship between protein sequence and structure, this work extends the knob‐socket model in an investigation of β‐sheet packing. Over a comprehensive set of β‐sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four‐residue packing cliques necessary to describe β‐sheet packing were characterized. Both occur between two adjacent hydrogen bonded β‐strands. First, defining the secondary structure packing within β‐sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β‐sheets, the knob‐socket XY:H+B consists of a three‐residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob‐sockets are found: side‐chain and main‐chain sockets. The amino acid composition of the pockets and knob‐sockets reveal the sequence specificity of β‐sheet packing. For β‐sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side‐chain and main‐chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β‐sheet structure and provide an intuitive topological mapping of β‐sheet packing. Proteins 2014; 82:2128–2140. © 2014 Wiley Periodicals, Inc.     

Slow formation of aggregation‐resistant β‐sheet folding intermediates

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding‐related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large β‐helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all‐β‐sheet protein allows detailed analysis of the formation of β‐sheet structure in larger proteins. Using a combination of fluorescence and far‐UV circular dichroism spectroscopy, we show that the pertactin β‐helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β‐sheet‐rich topology, pertactin refolding is reversible and not complicated by off‐pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate‐limiting step. Furthermore, site‐specific labeling experiments indicate that the β‐helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, β‐sheet‐rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β‐sheet‐rich refolding intermediates. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

VCD spectroscopic properties of the β‐hairpin forming miniprotein CLN025 in various solvents

Electronic and vibrational circular dichroism are often used to determine the secondary structure of proteins, because each secondary structure has a unique spectrum. Little is known about the vibrational circular dichroic spectroscopic features of the β‐hairpin. In this study, the VCD spectral features of a decapeptide, YYDPETGTWY (CLN025), which forms a stable β‐hairpin that is stabilized by intramolecular weakly polar interactions and hydrogen bonds were determined. Molecular dynamics simulations and ECD spectropolarimetry were used to confirm that CLN025 adopts a β‐hairpin in water, TFE, MeOH, and DMSO and to examine differences in the secondary structure, hydrogen bonds, and weakly polar interactions. CLN025 was synthesized by microwave‐assisted solid phase peptide synthesis with N^α‐Fmoc protected amino acids. The VCD spectra displayed a (?,+,?) pattern with bands at 1640 to 1656 cm^?1, 1667 to 1687 cm^?1, and 1679 to 1686 cm^?1 formed by the overlap of a lower frequency negative couplet and a higher frequency positive couplet. A maximum IR absorbance was observed at 1647 to 1663 cm^?1 with component bands at 1630 cm^?1, 1646 cm^?1, 1658 cm^?1, and 1675 to 1680 cm^?1 that are indicative of the β‐sheet, random meander, either random meander or loop and turn, respectively. These results are similar to the results of others, who examined the VCD spectra of β‐hairpins formed by ^DPro‐Xxx turns and indicated that observed pattern is typical of β‐hairpins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 442–450, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Hairpin formation promoted by the heterochiral dinipecotic acid segment: A DFT study

Conformational preferences for the turn and β‐hairpin structures of Ala‐based peptides [Ac‐Ala_n‐(R)‐Nip‐(S)‐Nip‐Ala_n‐X (n = 0–2; X = NHMe or NMe₂)] containing nipecotic acid (Nip) residues were carried out using the density functional M06‐2X and the implicit solvation model SMD in CH₂Cl₂ and/or water. The turn structure of the (R)‐Nip‐(S)‐Nip segment with a C₁₀ H‐bond between two terminal groups was found to be most preferred (populated at 98.9%) in CH₂Cl₂; this structure is consistent with IR and ¹H NMR results. The stabilities of the β‐hairpins containing the (R)‐Nip‐(S)‐Nip segment as a turn motif relative to the extended structures increased with peptide sequence length. The relative strengths of the H‐bonds between the carbonyl oxygen and the amide hydrogen appeared to be responsible for stabilizing the turn and β‐hairpin structures in CH₂Cl₂. In addition, the (R)‐Nip‐(S)‐Nip segment exhibited the capability to be incorporated into one of the two β‐turn motifs of gramicidin S (GS). The structure of this GS derivative (GS‐Nip₂) was generally similar to the native peptide but was less hydrophobic and it is therefore expected to exhibit lower hemolytic activity; however, further experiments are needed to evaluate its antimicrobial activity. The structure of GS‐Nip₂ was somewhat more flexible than GS in solvents of higher polarity. Thus, our calculated results regarding the turn and β‐hairpin motifs of the (R)‐Nip‐(S)‐Nip segment indicate that this structure might be useful for the design of bioactive macrocyclic peptides containing β‐hairpin mimics as well as binding epitopes in protein–protein and protein–nucleic acid recognitions. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 609–617, 2015.     

Helix‐sheet packing in proteins

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Although much is known about the packing geometry observed between α‐helices and between β‐sheets, there has been little progress on characterizing helix–sheet interactions. We present an analysis of the conformation of αβ₂ motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen bonded. The geometry of the αβ₂ motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β‐sheet. Side‐chain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ₂ motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native‐like conformations among a set of non‐native conformations in well‐known decoy datasets. The information on the geometry of αβ₂ motifs as well as the related statistical potentials have applications in the field of protein structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of the read-through domain from bacteriophage Qβ A1 protein

Bacteriophage Qβ is a small RNA virus that infects Escherichia coli. The virus particle contains a few copies of the minor coat protein A1, a C‐terminally prolonged version of the coat protein, which is formed when ribosomes occasionally read‐through the leaky stop codon of the coat protein. The crystal structure of the read‐through domain from bacteriophage Qβ A1 protein was determined at a resolution of 1.8 Å. The domain consists of a heavily deformed five‐stranded β‐barrel on one side of the protein and a β‐hairpin and a three‐stranded β‐sheet on the other. Several short helices and well‐ordered loops are also present throughout the protein. The N‐terminal part of the read‐through domain contains a prominent polyproline type II helix. The overall fold of the domain is not similar to any published structure in the Protein Data Bank.     

Propensities of peptides containing the Asn‐Gly segment to form β‐turn and β‐hairpin structures

The propensities of peptides that contain the Asn‐Gly segment to form β‐turn and β‐hairpin structures were explored using the density functional methods and the implicit solvation model in CH₂Cl₂ and water. The populations of preferred β‐turn structures varied depending on the sequence and solvent polarity. In solution, β‐hairpin structures with βI′ turn motifs were most preferred for the heptapeptides containing the Asn‐Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X‐ray structures. The sequence, H‐bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β‐hairpin structure of each heptapeptide, although the β‐turn segments played a role in promoting the formation of β‐hairpin structures and the β‐hairpin propensity varied. In the heptapeptides containing the Asn‐Gly segment, the β‐hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β‐turns and β‐hairpins containing the Asn‐Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β‐hairpin mimics and the design of binding epitopes for protein–protein and protein–nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653–664, 2016.     

An alternative structural isoform in amyloid‐like aggregates formed from thermally denatured human γD‐crystallin

The eye lens protein γD‐crystallin contributes to cataract formation in the lens. In vitro experiments show that γD‐crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV‐B photodamage results in its C‐terminal domain forming the β‐sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet‐like aggregates that contain cross‐linked oligomers of the protein, according to transmission electron microscopy and SDS‐PAGE. We use two‐dimensional infrared spectroscopy to show that these aggregates have an amyloid‐like secondary structure with extended β‐sheets, and use isotope dilution experiments to show that each protein contributes approximately one β‐strand to each β‐sheet in the aggregates. Using segmental ¹³C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N‐terminal and C‐terminal domains contribute to β‐sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N‐terminal domain into the fiber structure to intermolecular cross linking.     

Solution structure of protein synthesis initiation factor 1 from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation factor 1 from P. aeruginosa (Pa‐IF1) is the smallest of the three initiation factors that act to establish the 30S initiation complex during initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa‐IF1. Pa‐IF1 consists of a five‐stranded β‐sheet with an unusual extended β‐strand at the C‐terminus and one short α‐helix arranged in the sequential order β1‐β2‐β3‐α1‐β4‐β5. The structure adopts a typical β‐barrel fold and contains an oligomer‐binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α‐helix may compose the binding interface with the 30S subunit.     

Conformational analysis of the full‐length M2 protein of the influenza A virus using solid‐state NMR

The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N‐terminal ectodomain and the C‐terminal cytoplasmic tail remains largely unknown. Using two‐dimensional (2D) magic‐angle‐spinning solid‐state NMR, we have investigated the secondary structure and dynamics of full‐length M2 (M2FL) and found them to depend on the membrane composition. In 2D ¹³C DARR correlation spectra, 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)‐bound M2FL exhibits several peaks at β‐sheet chemical shifts, which result from water‐exposed extramembrane residues. In contrast, M2FL bound to cholesterol‐containing membranes gives predominantly α‐helical chemical shifts. Two‐dimensional J‐INADEQUATE spectra and variable‐temperature ¹³C spectra indicate that DMPC‐bound M2FL is highly dynamic while the cholesterol‐containing membranes significantly immobilize the protein at physiological temperature. Chemical‐shift prediction for various secondary‐structure models suggests that the β‐strand is located at the N‐terminus of the DMPC‐bound protein, while the cytoplasmic domain is unstructured. This prediction is confirmed by the 2D DARR spectrum of the ectodomain‐truncated M2(21–97), which no longer exhibits β‐sheet chemical shifts in the DMPC‐bound state. We propose that the M2 conformational change results from the influence of cholesterol, and the increased helicity of M2FL in cholesterol‐rich membranes may be relevant for M2 interaction with the matrix protein M1 during virus assembly and budding. The successful determination of the β‐strand location suggests that chemical‐shift prediction is a promising approach for obtaining structural information of disordered proteins before resonance assignment.     

Influence of ligand binding on structure and thermostability of human α1‐acid glycoprotein

Ligand binding of neutral progesterone, basic propranolol, and acidic warfarin to human α₁‐acid glycoprotein (AGP) was investigated by Raman spectroscopy. The binding itself is characterized by a uniform conformational shift in which a tryptophan residue is involved. Slight differences corresponding to different contacts of the individual ligands inside the β‐barrel are described. Results are compared with in silico ligand docking into the available crystal structure of deglycosylated AGP using quantum/molecular mechanics. Calculated binding energies are ?18.2, ?14.5, and ?11.5 kcal/mol for warfarin, propranolol, and progesterone, respectively. These calculations are consistent with Raman difference spectroscopy; nevertheless, minor discrepancies in the precise positions of the ligands point to structural differences between deglycosylated and native AGP. Thermal dynamics of AGP with/without bounded warfarin was followed by Raman spectroscopy in a temperature range of 10–95 °C and analyzed by principal component analysis. With increasing temperature, a slight decrease of α‐helical content is observed that coincides with an increase in β‐sheet content. Above 45 °C, also β‐strands tend to unfold, and the observed decrease in β‐sheet coincides with an increase of β‐turns accompanied by a conformational shift of the nearby disulfide bridge from high‐energy trans‐gauche‐trans to more relaxed gauche‐gauche‐trans. This major rearrangement in the vicinity of the bridge is not only characterized by unfolding of the β‐sheet but also by subsequent ligand release. Hereby, ligand binding alters the protein dynamics, and the more rigid protein–ligand complex shows an improved thermal stability, a finding that contributes to the reported chaperone‐like function of AGP. Copyright © 2015 John Wiley & Sons, Ltd.     

Solution structure for an Encephalitozoon cuniculi adrenodoxin‐like protein in the oxidized state

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria‐like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome‐like organelles in E. cuniculi, the 128‐residue, adrenodoxin‐like protein Ec‐Adx (UniProt ID Q8SV19) in the [2Fe‐2S] ferredoxin superfamily. Oxidized Ec‐Adx contains a mixed four‐strand β‐sheet, β2‐β1‐β4‐β3 (↓↑↑↓), loosely encircled by three α‐helices and two 3₁₀‐helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin‐like proteins except for the absence of a fifth anti‐parallel β‐strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the ¹H‐¹⁵N HSQC spectrum of Ec‐Adx. These missing residues are clustered primarily in two regions, G48‐V61 and L94‐L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe‐2S] cluster. Missing amide resonances in ¹H‐¹⁵N HSQC spectra are detrimental to NMR‐based solution structure calculations because ¹H‐¹H NOE restraints are absent (glass half‐empty) and this may account for the absent β‐strand (β5) and the position of α3 in oxidized Ec‐Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe‐2S] cluster in oxidized Ec‐Adx (glass half‐full).