Unusual Conformational Change Research Articles

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

A method for structure determination of GPCRs in various states.

G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the β2-adrenergic receptor (β2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For β2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structure‒function relationships and drug development.

Mechanism of activation and biased signaling in complement receptor C5aR1

The complement system plays an important role in the innate immune response to invading pathogens. The complement fragment C5a is one of its important effector components and exerts diverse physiological functions through activation of the C5a receptor 1 (C5aR1) and associated downstream G protein and β-arrestin signaling pathways. Dysfunction of the C5a-C5aR1 axis is linked to numerous inflammatory and immune-mediated diseases, but the structural basis for activation and biased signaling of C5aR1 remains elusive. Here, we present cryo-electron microscopy structures of the activated wild-type C5aR1–Gi protein complex bound to each of the following: C5a, the hexapeptidic agonist C5apep, and the G protein-biased agonist BM213. The structures reveal the landscape of the C5a–C5aR1 interaction as well as a common motif for the recognition of diverse orthosteric ligands. Moreover, combined with mutagenesis studies and cell-based pharmacological assays, we deciphered a framework for biased signaling using different peptide analogs and provided insight into the activation mechanism of C5aR1 by solving the structure of C5aR1I116A mutant–Gi signaling activation complex induced by C089, which exerts antagonism on wild-type C5aR1. In addition, unusual conformational changes in the intracellular end of transmembrane domain 7 and helix 8 upon agonist binding suggest a differential signal transduction process. Collectively, our study provides mechanistic understanding into the ligand recognition, biased signaling modulation, activation, and Gi protein coupling of C5aR1, which may facilitate the future design of therapeutic agents.

Physiological Metals Can Induce Conformational Changes in Transthyretin Structure: Neuroprotection or Misfolding Induction?

Transthyretin (TTR) is a plasma homotetrameric protein that transports thyroxine and retinol. TTR itself, under pathological conditions, dissociates into partially unfolded monomers that aggregate and form fibrils. Metal ions such as Zn2+, Cu2+, Fe2+, Mn2+ and Ca2+ play a controversial role in the TTR amyloidogenic pathway. TTR is also present in cerebrospinal fluid (CSF), where it behaves as one of the major Aβ-binding-proteins. The interaction between TTR and Aβ is stronger in the presence of high concentrations of Cu2+. Crystals of TTR, soaked in solutions of physiological metals such as Cu2+ and Fe2+, but not Mn2+, Zn2+, Fe3+, Al3+, Ni2+, revealed an unusual conformational change. Here, we investigate the effects that physiological metals have on TTR, in order to understand if metals can induce a specific and active conformation of TTR that guides its Aβ-scavenging role. The capability of certain metals to induce and accelerate its amyloidogenic process is also discussed.

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium.

Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Porcine kidney BADH (pkBADH) follows a bi-bi ordered mechanism in which NAD+ binds to the enzyme before the aldehyde. Previous studies showed that NAD+ induces complex and unusual conformational changes on pkBADH and that potassium is required to maintain its quaternary structure. The aim of this work was to analyze the structural changes in pkBADH caused by NAD+ binding and the role played by potassium in those changes. The pkBADH cDNA was cloned and overexpressed in Escherichia coli, and the protein was purified by affinity chromatography using a chitin matrix. The pkBADH/NAD+ interaction was analyzed by circular dichroism (CD) and by isothermal titration calorimetry (ITC) by titrating the enzyme with NAD+ . The cDNA has an open reading frame of 1485 bp and encodes a protein of 494 amino acids, with a predicted molecular mass of 53.9 kDa. CD data showed that the binding of NAD+ to the enzyme caused changes in its secondary structure, whereas the presence of K+ helps maintain its α-helix content. K+ increased the thermal stability of the pkBADH-NAD+ complex by 5.3°C. ITC data showed that NAD+ binding occurs with different association constants for each active site between 37.5 and 8.6 μM. All the results support previous data in which the enzyme incubation with NAD+ provoked changes in reactivity, which is an indication of slow conformational rearrangements of the active site.

An unusual conformational change in the folded trimethylene/leonard linker pyrazolo[3,4-d]pyrimidine analogue of the theophylline compound due to structural changes

The title compound C29H28N8O4, 1,1′-(propane-1,3-diyl)bis(5-benzyl-7-methyl-1H-pyrazolo[3,4-d]••pyrimidine-4,6(5H,7H)-dione) is characterized by single crystal X-ray diffraction. It crystallizes in the centrosymmetric space group P\(\bar 1\), with two molecules in the unit cell. There is one formula unit in the asymmetric unit with a folded conformation due to arene interaction and an unusually large angle of 124° in the propylene linker. The crystal structure is stabilized due to the presence of π–π and C–H…O interactions.

NMR Exchange Study of Z-DNA Duplexes Induced by the Zα Domain of Human ADAR1 Containing Mutation on the β-Hairpin

Left-handed Z-DNA forms in polymers of alternating d(CG)n sequence and is a higher energy conformation than B-DNA. Z-DNA is stabilized by high salt, negative supercoiling, and complex formation with Z-DNA binding proteins. The human double-stranded RNA deaminase I (ADAR1) contains two Z-DNA binding domains (Zα and Zβ) at the NH2-terminus. 4,5 The crystal structure of the Zα domain of human ADAR1 (ZαADAR1) complexed to Z-DNA showed that each of two Zα domains binds to one strand of a double-stranded DNA, yielding two-fold symmetry with respect to the DNA helical axis. This study also revealed that the intermolecular interaction is mediated by 5 residues in the α3 helix and 4 residues in the β-hairpin (Fig. 1). This is confirmed in solution by the significant backbone chemical shift changes of most residues in the α3 helix and β2-loop-β3 region upon binding to Z-DNA. These residues play important roles in Zα function and thus replacement of them with an alanine caused a dramatic decrease in Z-DNA binding affinity compared to wild-type ZαADAR1 (wt-ZαADAR1). 5 Previous NMR study suggested that single mutations at residues K169, N173, or Y177 cause unusual conformational changes in the hydrophobic faces of helices α1, α2, and α3, which dramatically decrease the Z-DNA binding affinity. This study also found that the K170A and R174A mutants significantly destabilize the G2·C5 base pair compared to wt-ZαADAR1, even though these mutants could efficiently change B-DNA to left-handed Z-DNA. However, the question of how the mutants on the β-hairpin affect the base pair stabilities of the Z-DNA remains. Here, we determined hydrogen exchange rate constants (kex) for the imino protons of the d(CG)3 in the complexes with ZαADAR1 containing mutation on the β-hairpin. Comparison of these results to previous studies of the mutant ZαADAR1-d(CG)3 complexes 8 provides the information required to identify the role of each residue in the β-hairpin during BZ transition of DNA duplexes.

Molecular structure of 1,2-bis(trifluoromethyl)-1,1,2,2-tetramethyldisilane in the gas, liquid, and solid phases: unusual conformational changes between phases.

The molecular structure of 1,2-bis(trifluoromethyl)-1,1,2,2-tetramethyldisilane has been determined in three different phases (solid, liquid, and gas) using various spectroscopic and diffraction techniques. Both the solid-state and gas-phase investigations revealed only one conformer to be present in the sample analyzed, whereas the liquid phase revealed the presence of three conformers. The data have been reproduced using computational methods and a rationale is presented for the observation of three conformers in the liquid state.

The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence.

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin–antitoxin proteins HipASO and HipBSO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin–antitoxin protein module. Here we show that phosphorylated HipASO can engage in an unexpected ternary complex with HipBSO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipBSO in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipASO in the ternary complex, indicative of its role in binding HipASO to abolish its function in persistence. The structure of HipASO in complex with a non-hydrolyzable ATP analogue shows that HipASO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipASO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.

NMR study of the Z-DNA binding mode and B–Z transition activity of the Zα domain of human ADAR1 when perturbed by mutation on the α3 helix and β-hairpin

The Zα domains of human ADAR1 (ZαADAR1) bind to Z-DNA via interaction mediated by the α3-core and β-hairpin. Five residues in the α3 helix and four residues in the β-hairpin play important roles in Zα function, forming direct or water-mediated hydrogen bonds with DNA backbone phosphates or interacting hydrophobically with DNA bases. To understand the roles of these residues during B–Z transition of duplex DNA, we performed NMR experiments on complexes of various ZαADAR1 mutants with a 6-bp DNA duplex at various protein-to-DNA molar ratios. Our study suggests that single mutations at residues K169, N173, or Y177 cause unusual conformational changes in the hydrophobic faces of helices α1, α2, and α3, which dramatically decrease the Z-DNA binding affinity. 1D imino proton spectra and chemical shift perturbation showed that single mutations at residues K170, R174, T191, P192, P193, or W195 slightly affected the Z-DNA binding affinity. A hydrogen exchange study proved that the K170A- and R174A-ZαADAR1 proteins could efficiently change B-DNA to left-handed Z-DNA via an active B–Z transition pathway, whereas the G2·C5 base pair was significantly destabilized compared to wild-type ZαADAR1.

Electrophoretic deposition of amphiphilic diacetylene supramolecules: polymerization, selective immobilization, pattern transfer and sensor applications

We describe the results of electrophoretic deposition characteristics of a conjugated polymer, polydiacetylene (PDA), that reveal film forming behavior, polymerization effects, pattern transfer capabilities, and fluorescence based thermo/mechano-responsive features of the deposited layer. The electric field applied in a two electrode system promotes the oriented motion of diacetylene (DA) supramolecules in aqueous solution in a direction that is determined by the charge polarities of tailorable pendant groups on the DA monomers. Through this mechanism, electrophoretic deposition of DA supramolecules on a conductive layer takes place to yield vesicular clustered aggregates with continuous leaf-like morphologies. In addition, unusual conformational changes of the DA monomers during the deposition result in the formation of polymerized blue colored PDAs on the working electrode. Based on these properties, selective deposition of the supramolecules on pre-patterned templates and consequent pattern transfer to a flexible substrate can be utilized to fabricate PDA-based microarray sensors. The chromatic change of the vesicles induced by an applied mechanical stress during pattern transfer is found to be dependent on the side groups of the DA monomers. Owing to inherent non-fluorescence to fluorescence transition features of PDA supramolecules occurring as a response to thermal perturbations, electrophoretically deposited PDA vesicles are employed to monitor the temperature of a heat dispensing micro-heater device.

Structure of the apo decarbamylated form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase fromBacillus subtilis

2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase), a RuBisCO-like protein (RLP), catalyzes the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate. The crystal structure of the apo decarbamylated form (E form) of Bacillus subtilis DK-MTP-1P enolase (Bs-DK-MTP-1P enolase) has been determined at 2.3 A resolution. The overall structure of the E form of Bs-DK-MTP-1P enolase highly resembles that of Geobacillus kaustophilus DK-MTP-1P enolase (Gk-DK-MTP-1P enolase), with the exception of a few insertions or deletions and of a few residues at the active site. In the E form of Bs-DK-MTP-1P enolase, Lys150 (equivalent to Lys175 in RuBisCO) at the active site adopts a conformation that is distinct from those observed in the other forms of Gk-DK-MTP-1P enolase. This unusual conformational change appears to be induced by changes in the varphi and psi angles of Gly151, which is conserved in the sequences of the Bs-DK-MTP-1P and Gk-DK-MTP-1P enolases but not in those of RuBisCOs. The loop at 303-312, equivalent to the catalytic loop termed ;loop-6' in RuBisCO, is in a closed conformation in the E form of Bs-DK-MTP-1P enolase. The closed conformation appears to be stabilized by Pro312, which is conserved in the sequences of several RLPs (equivalent to Glu338 in RuBisCO). Based on these results, the characteristic structural features of DK-MTP-1P enolase are discussed.

Correlated Motions and Interactions at the Onset of the DNA-Induced Partial Unfolding of Ets-1

The binding of the Ets-1 transcription factor to its target DNA sequence is characterized by a highly unusual conformational change consisting of the unfolding of inhibitory helix 1 (HI-1). To probe the interactions that lead to this unfolding, we performed molecular dynamics simulations of the folded states of apo-Ets-1 and the Ets-1-DNA complex. The simulations showed large differences in correlated motions between helix 4 (H4) and HI-1. In apo-Ets-1, H4 and HI-1 moved in-phase and stabilized each other by hydrogen bonding and macrodipolar interactions, whereas in the DNA-bound state, the motion was out-of-phase, with a disruption of the stabilizing interactions. This change in motion was due to hydrogen-bonding interactions between helix 1 (H1) and the DNA. The dipolar energy between H1 and H4 was modulated by hydrogen bonds between H1 and DNA, and, in accordance with experiments, elimination of the hydrogen bonds increased the stability of HI-1. The simulations confirm that the hydrogen bonds between H1 and DNA act as a conformational switch and show that the presence of DNA is communicated from H1 to H4, destabilizing HI-1. The calculations reveal a critical role for correlated motions at the onset of the DNA-induced unfolding.

Identification and characterization of a novel fibril forming peptide in fungal starch binding domain

Scanty information is available regarding the chemical basis for structural alterations of the carbohydrate-binding modules (CBMs). The N-terminal starch binding domain (SBD) of Rhizopus oryzae glucoamylase (GA) forms fibrils under thermal stress, presenting an unusual conformational change from immunoglobulin-like to β-sheet-rich structure. Site-directed mutagenesis revealed that the C-terminal Lys of SBD played a crucial role in the fibril formation. The synthetic peptide (DNNNSANYQVSTSK) representing the C-terminal 14 amino acid residues of SBD was further demonstrated to act as a fibril-forming segment, in which terminal charges and an internal NNNxxNYQ motif were key fibril-forming determinants. The formation of fibril structure in a fungal SBD, caused by its chemical and biophysical requirements, was demonstrated for the first time.

Complex, unusual conformational changes in kidney betaine aldehyde dehydrogenase suggested by chemical modification with disulfiram

The NAD +-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD + protected almost 100% at 10 μM but only 50% at 5 mM, and vice versa if the enzyme was pre-incubated with NAD + before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import.

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.

A SOX9 Defect of Calmodulin-dependent Nuclear Import in Campomelic Dysplasia/Autosomal Sex Reversal

During mammalian sex determination, SOX9 is translocated into the nuclei of Sertoli cells within the developing XY gonad. The N-terminal nuclear localization signal (NLS) is contained within a SOX consensus calmodulin (CaM) binding region, thereby implicating CaM in nuclear import of SOX9. By fluorescence spectroscopy and glutaraldehyde cross-linking, we show that the SOX9 HMG domain and CaM interact in vitro. The formation of a SOX9.CaM binary complex is calcium-dependent and is accompanied by a conformational change in SOX9. A CaM antagonist, calmidazolium chloride (CDZ), was observed to block CaM recognition of SOX9 in vitro and inhibit both nuclear import and consequent transcriptional activity of SOX9 in treated cells. The significance of the SOX9-CaM interaction was highlighted by analysis of a missense SOX9 mutation, A158T, identified from a XY female with campomelic dysplasia/autosomal sex reversal (CD/SRA). This mutant binds importin beta normally despite defective nuclear import. Fluorescence and quenching studies indicate that in the unbound state, the A158T mutant shows a similar conformation to that of the WT SOX9, but in the presence of CaM, the mutant undergoes unusual conformational changes. Furthermore, SOX9-mediated transcriptional activation by cells expressing the A158T mutant is more sensitive to CDZ than cells expressing WT SOX9. These results suggest first that CaM is involved in the nuclear transport of SOX9 in a process likely to involve direct interaction and second, that CD/SRA can arise, at least in part, from a defect in CaM recognition, ultimately leading to reduced ability of SOX9 to activate transcription of cartilage and testes-forming genes.

Unusual Conformational Changes in 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase as Revealed by X-ray Crystallography and NMR

The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle.

Efficient intermolecular energy transfer between soluble polysilanes

We observed an intermolecular energy transfer from polydihexylsilane (PDHS) to poly bis( p-propoxyphenyl)silane (PBPPS) in both PDHS/PBPPS double layer and blend films by utilizing photoluminescence (PL) spectroscopy for the first time. Decrease in the intensity of PDHS emission and increase in that of PBPPS emission occurred in the PDHS/PBPPS double layer film. In case of the PDHS/PBPPS blend film, unusual conformational change in the backbone of PDHS was observed due to the co-existence of PBPPS molecules. The PL excitation spectra and time resolved PL measurement indicate that the resonant energy transfer process dominates the entire transfer process.

Human plasminogen catalytic domain undergoes an unusual conformational change upon activation

Activation of the serine protease plasmin from its zymogen, plasminogen, is the key step in fibrinolysis leading to blood clot dissolution. It also plays critical roles in cell migration, such as in tumor metastasis. Here, we report the crystal structure of an inactive S741A mutant of human plasminogen catalytic domain at 2.0 Å resolution. This structure permits a direct comparison with that of the plasmin catalytic unit. Unique conformational differences are present between these two structures that are not seen in other zymogen-enzyme pairs of the trypsin family. The functional significance of these differences and the structural basis of plasminogen activation is discussed in the light of this new structure.