A Hinge Region cis-Proline in Ribonuclease A Acts as a Conformational Gatekeeper for C-Terminal Domain Swapping

Domain swapping, the process in which a structural unit is exchanged between monomers to create a dimer containing two subunits of the monomeric fold, is believed to be an important mechanism for oligomerization and the formation of amyloid fibrils. Structural studies have implicated proline as an important residue for domain swapping due to its increased frequency in hinge regions preceding swapped arms. We hypothesized that proline's unique ability to populate both cis and trans peptide bond conformations may allow proline to act as a conformational gatekeeper, regulating interconversion between monomer and domain-swapped dimer forms. The hinge region of RNase A contains a proline at residue 114 that adopts a cis conformation in the monomer and extends to a trans conformation in the C-terminal domain-swapped dimer. Substitution of P114 with residues that strongly prefer a trans peptide bond (Ala, Gly) results in significant population of the C-terminal domain-swapped dimer under near-physiological conditions (pH 8.0, 37 °C). This is in stark contrast to dimerization of wild-type RNase A, which requires incubation under extreme conditions such as lyophilization from acetic acid or elevated temperature. In addition, we observe similar results when cis-P114 is mutated to glycine in a homologous RNase, human pancreatic RNase 1. Our results suggest that isomerization at P114 may facilitate population of a partially unfolded intermediate or alternative structure competent for domain swapping and provide support for a more general role for proline isomerization as a conformational gatekeeper in domain swapping and oligomerization.     

Characterization of 14-3-3sigma Dimerization Determinants: Requirement of Homodimerization for Inhibition of Cell Proliferation

The seven highly conserved 14-3-3 proteins expressed in mammalian cells form a complex pattern of homo- and hetero-dimers, which is poorly characterized. Among the 14-3-3 proteins 14-3-3sigma is unique as it has tumor suppressive properties. Expression of 14-3-3sigma is induced by DNA damage in a p53-dependent manner and mediates a cell cycle arrest. Here we show that the 14-3-3sigma protein exclusively forms homodimers when it is ectopically expressed at high levels, whereas ectopic 14-3-3zeta formed heterodimers with the 5 other 14-3-3 isoforms. The x-ray structure of 14-3-3sigma?revealed 5 residues (Ser⁵, Glu²⁰, Phe²⁵, Q⁵⁵, Glu⁸⁰) as candidate determinants of dimerization specificity. Here we converted these amino-acids to residues present in 14-3-3zeta at the analogous positions. Thereby, Ser⁵, Glu²⁰ and Glu⁸⁰ were identified as key residues responsible for the selective homodimerization of 14-3-3sigma. Conversion of all 5 candidate residues was sufficient to switch the dimerization pattern of 14-3-3sigma to a pattern which is very similar to that of 14-3-3zeta. In contrast to wildtype 14-3-3sigma this 14-3-3sigma variant and 14-3-3zeta were unable to mediate inhibition of cell proliferation. Therefore, homodimerization by 14-3-3sigma is required for its unique functions among the 7 mammalian 14-3-3 proteins. As inactivation of 14-3-3sigma sensitizes to DNA-damaging drugs, substances designed to interfere with 14-3-3sigma dimerization may be used to inactivate 14-3-3sigma function for cancer therapeutic purposes.     

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His²⁸¹ (A1 domain) with Ser⁵²⁴ (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His²⁸¹ and Ser⁵²⁴ residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, K_m for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His²⁸¹ and Ser⁵²⁴ are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.     

Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled α-Factor Receptor

The α-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the α-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the α-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser²⁵⁴ showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln²⁵³ and Ser²⁵⁴ are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln²⁵³ and Ser²⁵⁴ fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln²⁵³ and Ser²⁵⁴ face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser²⁸⁸ and Ser²⁹²) that are potential contact residues for Gln²⁵³ because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the α-factor receptor that regulates its ability to enter the activated conformation.     

In silico phosphorylation of the autoinhibited form of p47(phox): insights into the mechanism of activation

Activation of the multicomponent enzyme NADPH oxidase requires the interaction between the tandem SH3 domain of the cytosolic subunit p47^phox and the cytoplasmic tail of membrane-bound p22^phox. In the resting state, p47^phox exists in an autoinhibited conformation stabilized by intramolecular contacts between the SH3 domains and an adjacent polybasic region. Phosphorylation of three serine residues, Ser³⁰³, Ser³⁰⁴, and Ser³²⁸ within this polybasic region has been shown to be sufficient for the disruption of the intramolecular interactions thereby inducing an active state of p47^phox. This active conformation is accessible to the cytoplasmic tail of p22^phox and initiates the formation of the membrane-bound functional enzyme complex. Molecular dynamics simulations reveal insights in the mechanism of activation of the autoinhibited form of p47^phox by in silico phosphorylation, of the three serine residues, Ser³⁰³, Ser³⁰⁴, and Ser³²⁸. The simulations highlight the major collective coordinates generating the opening and the closing of the two SH3 domains and the residues that cause the unmasking of the p22^phox binding site.     

Biophysical Analysis of the MHR Motif in Folding and Domain Swapping of the HIV Capsid Protein C-Terminal Domain

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.     

The structure of an engineered domain-swapped ribonuclease dimer and its implications for the evolution of proteins toward oligomerization

BACKGROUND: Domain swapping has been proposed as a mechanism that explains the evolution from monomeric to oligomeric proteins. Bovine and human pancreatic ribonucleases are monomers with no biological properties other than their RNA cleavage ability. In contrast, the closely related bovine seminal ribonuclease is a natural domain-swapped dimer that has special biological properties, such as cytotoxicity to tumour cells. Several recombinant ribonuclease variants are domain-swapped dimers, but a structure of this kind has not yet been reported for the human enzyme. RESULTS: The crystal structure at 2 A resolution of an engineered ribonuclease variant called PM8 reveals a new kind of domain-swapped dimer, based on the change of N-terminal domains between the two subunits. The swapping is fastened at both hinge peptides by the newly introduced Gln101, involved in two intermolecular hydrogen bonds and in a stacking interaction between residues of different chains. Two antiparallel salt bridges and water-mediated hydrogen bonds complete a new interface between subunits, while the hinge loop becomes organized in a 3(10) helix structure. CONCLUSIONS: Proteins capable of domain swapping may quickly evolve toward an oligomeric form. As shown in the present structure, a single residue substitution reinforces the quaternary structure by forming an open interface. An evolutionary advantage derived from the new oligomeric state will fix the mutation and favour others, leading to a more extended complementary dimerization surface, until domain swapping is no longer necessary for dimer formation. The newly engineered swapped dimer reported here follows this hypothetical pathway for the rapid evolution of proteins.     

Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase.

Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.     

Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease

Background

A highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and other places in Saudi Arabia, and has quickly spread to European and Asian countries since September 2012. Up to the 1^st October 2015 it has infected at least 1593 people with a global fatality rate of about 35%. Studies to understand the virus are necessary and urgent. In the present study, MERS-CoV main protease (M^pro) is expressed; the dimerization of the protein and its relationship to catalysis are investigated.

Methods and Results

The crystal structure of MERS-CoV M^pro indicates that it shares a similar scaffold to that of other coronaviral M^pro and consists of chymotrypsin-like domains I and II and a helical domain III of five helices. Analytical ultracentrifugation analysis demonstrated that MERS-CoV M^pro undergoes a monomer to dimer conversion in the presence of a peptide substrate. Glu169 is a key residue and plays a dual role in both dimerization and catalysis. The mutagenesis of other residues found on the dimerization interface indicate that dimerization of MERS-CoV M^pro is required for its catalytic activity. One mutation, M298R, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme.

Conclusions

MERS-CoV M^pro shows substrate-induced dimerization and potent proteolytic activity. A critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral M^pro family.     

Metal ion determinants of conantokin dimerization as revealed in the X-ray crystallographic structure of the Cd2+/Mg2+–con-T[K7γ] complex

Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in γ-carboxyglutamic acid (Gla) residues. As potent and selective antagonists of the N-methyl-d-aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10, and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca²⁺ and Mg²⁺ can fulfill this role, Ca²⁺ induces dimerization of con-G, whereas the Mg²⁺-complexed peptide remains monomeric. A variant of con-T, con-T[K7γ] (γ is Gla), contains Gla residues at the same five positions as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca²⁺ ions and two Mg²⁺ ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca²⁺ for dimer formation, we report here the structure of the monomeric Cd²⁺/Mg²⁺–con-T[K7γ] complex, and, by comparison with the previously published con-T[K7γ]/Ca²⁺ dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization.     

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.     

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation

The neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process. Here, we retrieved experimentally observed phosphorylation sites of KCC2 from public databases and report on the systematic analysis of six phosphorylated serines, Ser²⁵, Ser²⁶, Ser⁹³⁷, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶. Alanine or aspartate substitutions of these residues were analyzed in HEK-293 cells. All mutants were expressed in a pattern similar to wild-type KCC2 (KCC2^WT). Tl⁺ flux measurements demonstrated unchanged transport activity for Ser²⁵, Ser²⁶, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶ mutants. In contrast, KCC2^S937D, mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr⁹³⁴, a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2^T934D and KCC2^S937D mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2^WT, KCC2^T934A, and KCC2^S937A were activated. The inverse staurosporine effect on aspartate versus alanine substitutions reveals a cross-talk between different phosphorylation sites of KCC2. Immunoblot and cell surface labeling experiments detected no alterations in total abundance or surface expression of KCC2^T934D and KCC2^S937D compared with KCC2^WT. These data reveal kinetic regulation of transport activity by these residues. In summary, our data identify a novel key regulatory phosphorylation site of KCC2 and a functional interaction between different conformation-changing post-translational modifications. The action of pharmacological agents aimed to modulate KCC2 activity for therapeutic benefit might therefore be highly context-specific.     

A comprehensive comparison of the metazoan tryptophan degrading enzymes

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) have an independent origin; however, they have distinctly evolved to catalyze the same reaction. In general, TDO is a single-copy gene in each metazoan species, and TDO enzymes demonstrate similar enzyme activity regardless of their biological origin. In contrast, multiple IDO paralogues are observed in many species, and they display various enzymatic properties. Similar to vertebrate IDO2, invertebrate IDOs generally show low affinity/catalytic efficiency for L-Trp. Meanwhile, two IDO isoforms from scallop (IDO-I and -III) and sponge IDOs show high L-Trp catalytic activity, which is comparable to vertebrate IDO1. Site-directed mutagenesis experiments have revealed that primarily two residues, Tyr located at the 2nd residue on the F-helix (F2^nd) and His located at the 9th residue on the G-helix (G9^th), are crucial for the high affinity/catalytic efficiency of these ‘high performance’ invertebrate IDOs. Conversely, those two amino acid substitutions (F2^nd/Tyr and G9^th/His) resulted in high affinity and catalytic activity in other molluscan ‘low performance’ IDOs. In human IDO1, G9^th is Ser¹⁶⁷, whereas the counterpart residue of G9^th in human TDO is His⁷⁶. Previous studies have shown that Ser¹⁶⁷ could not be substituted by His because the human IDO1 Ser¹⁶⁷His variant showed significantly low catalytic activity. However, this may be specific for human IDO1 because G9^th/His was demonstrated to be very effective in increasing the L-Trp affinity even in vertebrate IDOs. Therefore, these findings indicate that the active sites of TDO and IDO are more similar to each other than previously expected.     

SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped β-Sandwich Fold Important for SOCE Modulation

Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca²⁺ stores and generate intracellular Ca²⁺ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X‐ray crystal structure of the SARAF luminal domain, SARAF_L. This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the “SARAF-fold.” The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the “SARAF luminal switch” that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation.     

Structural Insight into Substrate Differentiation of the Sugar-metabolizing Enzyme Galactitol Dehydrogenase from Rhodobacter sphaeroides D

Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo- and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn¹¹⁶, Ser¹⁴⁴, Tyr¹⁵⁹, and Lys¹⁶³. GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser¹⁴⁴, Ser¹⁴⁶, and Asn¹⁵¹ are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.     

Detection of two CSN1S1 variants in Egyptian buffalo

The present study investigates CSN1S1 casein gene polymorphism in Egyptian buffalo. CSN1S1 was analyzed in 17 unrelated Egyptian lactating buffalo. The amplified segment includes the last 43 amino acids of Exon 17 and part of Intron 17. In the present study we report for the first time the presence of 2 variants 178^Ser (TCA) and 178^Leu (TTA) in Egyptian buffalo CSN1S1 gene. The genotypic frequencies in the investigated Egyptian buffalo sample were 0.47, 0.058 and 0.47 for homozygous 178^Ser, for homozygous 178^Leu and heterozygous 178^Leu/Ser, respectively. The 178^Ser and 178^Leu variant frequencies are 0.64 and 0.36, respectively which indicates the superiority of variant 178^Ser in Egyptian buffalo. The allelic frequency in Egyptian buffalo is not much different from the corresponding allelic frequency in Italian buffalo (0.69 and 0.31 for 178^Ser and 178^Leu, respectively) as reported by Chianese et al. [3]. This is not surprising since they both belong to Mediterranean type.     

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

The dimerization of ferrihaems: III. Equilibrium and kinetic studies on the dimerization of coproferrihaem

Spectrophotometric studies on the behaviour of coproferrihaem in aqueous solution showed that, in the pH range 6.66–8.04, a dimerization process occurs according to the equation 2 monomer

The value of K, the pH-independent dimerization contant, was found to be 2.10 · 10^?3, signifying that coproferrihaem shows the least tendency to dimerize of any ferrihaem so far investigated. Forward and reverse rate constants for the dimerization process have been determined by the temperature-jump method.The results suggest that the cation-briding between carboxyl residues, postulated for the dimers of the dicarboxylic ferrihaems, cannot occur between the additional carboxyl residues of coproferrihaem and that the increased negative charge may cause destabilization of the coproferrihaem dimer by repulsion effects.     

Stability strengths and weaknesses in protein structures detected by statistical potentials: Application to bovine seminal ribonuclease

We present an in silico method to estimate the contribution of each residue in a protein to its overall stability using three database‐derived statistical potentials that are based on inter‐residue distances, backbone torsion angles and solvent accessibility, respectively. Residues that contribute very unfavorably to the folding free energy are defined as stability weaknesses, whereas residues that show a highly stabilizing contribution are called stability strengths. Strengths and/or weaknesses on residues that are in spatial contact are clustered into 3‐dimensional (3D) stability patches. The identification and analysis of strength‐ and weakness‐containing regions in a protein may reveal structural or functional characteristics, and/or interesting spots to introduce mutations. To illustrate the power of our method, we apply it to bovine seminal ribonuclease. This enzyme catalyzes the degradation of RNA strands, and has the peculiarity of undergoing 3D domain swapping in physiological conditions. The weaknesses and strengths were compared among the monomeric, dimeric and swapped dimeric forms. We identified weaknesses among the catalytic residues and a mixture of weaknesses and strengths among the substrate‐binding residues in the three forms. In the regions involved in 3D swapping, we observed an accumulation of weaknesses in the monomer, which disappear in the dimer and especially in the swapped dimer. Moreover, monomeric homologous proteins were found to exhibit less weaknesses in these regions, whereas mutants known to favor unswapped dimerization appear stabilized in this form. Our method has several perspectives for functional annotation, rational prediction of targeted mutations, and mapping of stability changes upon conformational rearrangements. Proteins 2016; 84:143–158. © 2015 Wiley Periodicals, Inc.     

Interdependent Phosphorylation within the Kinase Domain T-loop Regulates CHK2 Activity

Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser¹⁹, Ser³³, Ser³⁵, and Thr⁶⁸. This precedes autophosphorylation of Thr³⁸³/Thr³⁸⁷ in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366–406). We report four novel phosphorylation sites at Ser³⁷², Thr³⁷⁸, Thr³⁸⁹, and Tyr³⁹⁰. Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr³⁸⁹ was a prerequisite to phosphorylation at Thr³⁸⁷ but not at Thr³⁸³. Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser³⁷⁹, Thr³⁸⁹, and Thr³⁸³/Thr³⁸⁹. Phosphorylation at Tyr³⁹⁰ was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr³⁷², Thr³⁷⁹, Thr³⁸³, Thr³⁸⁹, Thr³⁸³/Thr³⁸⁷, and Thr³⁸³/Thr³⁸⁹. Chk2 hyper-phosphorylated species at Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹ and Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹/Tyr³⁹⁰ relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.