Serpin crystal structure and serpin polymer structure

Serpins are a family of structurally homologous proteins having metastable native structures. As a result, a serpin variant destabilized by mutation(s) has a tendency to undergo conformational changes leading to inactive forms, e.g., the latent form and polymer. Serpin polymers are involved in a number of conformational diseases. Although several models for polymer structure have been proposed, the actual structure remains unknown. Here, we provide a comprehensive list of serpins, both free and in complexes, deposited in the Protein Data Bank. Our discussion focuses on structures that potentially can contribute to a better understanding of polymer structure.     

Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes

Pontin and reptin belong to the AAA+ family, and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 A. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared with the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different than previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins.     

1H and 13C-NMR and molecular dynamics studies of cyclosporin a interacting with magnesium(II) or cerium(III) in acetonitrile. Conformational changes and cis-trans conversion of peptide bonds

Cyclosporin A (CsA) is an important drug used to prevent graft rejection in organ transplantations. Its immunosuppressive activity is related to the inhibition of T-cell activation through binding with the proteins Cyclophilin (Cyp) and, subsequently, Calcineurin (CN). In the complex with its target (Cyp), CsA adopts a conformation with all trans peptide bonds and this feature is very important for its pharmacological action. Unfortunately, CsA can cause several side effects, and it can favor the excretion of calcium and magnesium. To evaluate the possible role of conformational effects induced by these two metal ions in the action mechanism of CsA, its complexes with Mg(II) and Ce(III) (the latter as a paramagnetic probe for calcium) have been examined by two-dimensional NMR and relaxation rate analysis. The conformations of the two complexes and of the free form have been determined by restrained molecular dynamics calculations based on the experimentally obtained metal-proton and interproton distances. The findings here ratify the formation of 1:1 complexes of CsA with both Mg(II) and Ce(III), with metal coordination taking place on carbonyl oxygens and substantially altering the peptide structure with respect to the free form, although the residues involved and the resulting conformational changes, including cis-trans conversion of peptide bonds, are different for the two metals.     

Effect of cytochrome b5 on the functional activity and conformational state of cytochrome P-450

Microsomal cytochromes P-450 and b5 were shown to form mixed complexes with the association constant of 0.24 microM in water solution. Such complex formation stabilizes cytochrome P-450 in the catalytically active conformational state characterized by increased conformational rigidity and temperature stability. This stabilization results in acceleration of the cumene hydroperoxide-dependent oxidation of p-nitroanisol catalyzed by cytochrome P-450. The thermodynamic parameters of O-demethylation of p-nitroanisol catalyzed by cytochrome P-450 and mixed haemoprotein complexes measured in water solution and in a membrane-bound state were found to be different.     

Pre- and post-docking sampling of conformational changes using ClustENM and HADDOCK for protein-protein and protein-DNA systems

Incorporating the dynamic nature of biomolecules in the modeling of their complexes is a challenge, especially when the extent and direction of the conformational changes taking place upon binding is unknown. Estimating whether the binding of a biomolecule to its partner(s) occurs in a conformational state accessible to its unbound form (“conformational selection”) and/or the binding process induces conformational changes (“induced-fit”) is another challenge. We propose here a method combining conformational sampling using ClustENM—an elastic network-based modeling procedure—with docking using HADDOCK, in a framework that incorporates conformational selection and induced-fit effects upon binding. The extent of the applied deformation is estimated from its energetical costs, inspired from mechanical tensile testing on materials. We applied our pre- and post-docking sampling of conformational changes to the flexible multidomain protein-protein docking benchmark and a subset of the protein-DNA docking benchmark. Our ClustENM-HADDOCK approach produced acceptable to medium quality models in 7/11 and 5/6 cases for the protein-protein and protein-DNA complexes, respectively. The conformational selection (sampling prior to docking) has the highest impact on the quality of the docked models for the protein-protein complexes. The induced-fit stage of the pipeline (post-sampling), however, improved the quality of the final models for the protein-DNA complexes. Compared to previously described strategies to handle conformational changes, ClustENM-HADDOCK performs better than two-body docking in protein-protein cases but worse than a flexible multidomain docking approach. However, it does show a better or similar performance compared to previous protein-DNA docking approaches, which makes it a suitable alternative.     

Structural diversity of human class II histocompatibility molecules induced by peptide ligands

SDS-PAGE analyses of stable HLA-DR1 complexes indicate that the binding of T cell epitopes can lead to multiple conformational variants. Whereas short T epitopes (<14-mer) induce complexes with apparent MW ranging from 47 to 57 kDa, longer peptides form generally high mobility complexes (44-45 kDa). The generation of HLA-DR1 conformational variants appears dependent on core peptide residues fitting inside the groove but can additionally be attributed to the presence of N- and C-terminal flanking residues (PFRs) acting as a complementary mechanism. These PFRs can jointly affect major histocompatibility complex class II conformation and stability, supporting the existence of alternative contacts at a distance from the classical binding site.     

Structural Plasticity and Conformational Transitions of HIV Envelope Glycoprotein gp120

HIV envelope glycoproteins undergo large-scale conformational changes as they interact with cellular receptors to cause the fusion of viral and cellular membranes that permits viral entry to infect targeted cells. Conformational dynamics in HIV gp120 are also important in masking conserved receptor epitopes from being detected for effective neutralization by the human immune system. Crystal structures of HIV gp120 and its complexes with receptors and antibody fragments provide high-resolution pictures of selected conformational states accessible to gp120. Here we describe systematic computational analyses of HIV gp120 plasticity in such complexes with CD4 binding fragments, CD4 mimetic proteins, and various antibody fragments. We used three computational approaches: an isotropic elastic network analysis of conformational plasticity, a full atomic normal mode analysis, and simulation of conformational transitions with our coarse-grained virtual atom molecular mechanics (VAMM) potential function. We observe collective sub-domain motions about hinge points that coordinate those motions, correlated local fluctuations at the interfacial cavity formed when gp120 binds to CD4, and concerted changes in structural elements that form at the CD4 interface during large-scale conformational transitions to the CD4-bound state from the deformed states of gp120 in certain antibody complexes.     

Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin

These studies explore the role of conformational change and exposed carbohydrate residues in the clearance of alpha 2-macroglobulin-trypsin (alpha 2M-T) complexes in the mouse. Human alpha 2-macroglobulin (alpha 2M) was purified and demonstrated to be homogeneous in the electrophoretic "slow" form. Two conformationally altered derivatives, alpha 2M-T and alpha 2-macroglobulin-methylamine (alpha 2M-MeNH2), were prepared and demonstrated to exist in the electrophoretic "fast" form. Radiolabeled alpha 2M-T and alpha 2M-MeNH2 were cleared rapidly with a half-life of 2-4 min following injection into mice. Radiolabeled native alpha 2M, however, remained in the circulation with a half-life of several hours. Both alpha 2M-T and alpha 2M-MeNH2 bound specifically to mouse peritoneal macrophages at 4 degrees C and occupancy of receptor sites increased with increasing time and radioligand concentration. Excess amounts of unlabeled alpha 2M-T or alpha 2M-MeNH2 cross-completed with trace amounts of the other in both clearance studies and binding assays, indicating that both derivatives were removed by the same receptor pathway. The clearance and binding of alpha 2M-T and alpha 2M-MeNH2 were not inhibited by excess amounts of unlabeled asialoorosomucoid, fucosyl-bovine serum albumin, mannosyl-BSA, or N-acetylglucosaminyl-BSA. Our results indicate that the clearance pathway removing alpha 2M-T complexes from the circulation recognizes a fundamental conformational change in alpha 2M secondary to protease binding, which can also be induced by exposure to methylamine. Therefore, other chemical or physical alterations that occur in alpha 2M upon binding trypsin, apart from the conformational change also present in alpha 2M-MeNH2, do not seem necessary for the recognition of alpha 2M-T by cells in the clearance pathway. In addition, this pathway appears distinct from several systems already described mediating clearance of glycoproteins through recognition of terminal galactose, fucose, N-acetylglucosamine, or mannose on oligosaccharide side chains.     

13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid.

13C NMR studies of 13C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-13C]- and [2,4a,6-13C]methotrexate (MTX) and [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180 degrees difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR-MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.     

Solution conformations of valinomycin-divalent cation complexes

The solution conformations of complexes of valinomycin with magnesium and strontium were investigated by circular dichroism, nuclear magnetic resonance and infrared techniques. The results were compared with our earlier results on lithium, calcium, manganese and barium complexes. All these cations, except lithium, form 2:1 ion sandwich and 1:1 carrier-cation complexes with valinomycin. The 1:1 complex has a conformation different from that of the valinomycin-potassium complex. Lithium forms only the 1:1 complex. Strontium and barium form a large number of 1:2 complexes with open conformations rapidly interconverting in solution in addition to the above 2:1 and 1:1 complexes. These observations are rationalized taking into account the ionic radii and coordination numbers of the cations and the conformational restraints of valinomycin molecules. It is suggested that cations with co-ordination numbers of about six (magnesium and calcium) form the 2:1 and 1:1 complexes whereas those with higher co-ordination numbers (strontium and barium) form 1:2 complexes also.     

Studies of enzyme-ligand complexes using dynamic fluorescence anisotropy. II. The coenzyme-binding site of malate dehydrogenase

The coenzyme-binding site in mitochondrial malate dehydrogenase from pig heart was studied using dynamic fluorescence anisotropy decay. The dynamics of the fluorescent ligands NADH and 6-cyano-7-hydroxy-4,8-dimethylcoumarin were used to detect conformational changes at the dihydronicotinamide-and at the adenosine-binding sites, respectively. Addition of the natural substrate L-malate to the complex from enzyme and NADH does not influence the complete immobilization of the dihydronicotinamide group, whereas the stereoisomer D-malate and the substrate-analogue hydroxymalonate form ternary complexes with highly mobile dihydronicotinamide. The dynamics of the fluorescent adenosine-analogue are not influenced by formation of complexes with substrate and substrate-analogues. Thus the conformational changes at the dihydronicotinamide-binding site remain local and are not transmitted to the adenosine-binding site.     

Interaction of electron acceptors with the excited triplet state of Zn cytochrome c

The decay rate of the excited triplet state of Zn cytochrome c was enhanced by electron acceptors including methyl viologen and ferric complexes of cyanide, oxalate, EDTA and cytochrome c at room temperature. Ferrous compounds were several orders of magnitude less effective than the respective ferric form in quenching the phosphorescence. In the presence of ferricytochrome c and ferricyanide the semilogarithmic plots of the decay curve showed an anomalous decay profile in which the rate of interaction appeared to accelerate after excitation. One explanation is that the quenching process was accelerated by a conformational change of the polypeptide chain around the excited triplet state porphyrin. Another explanation is that quenching occurs via an intermediate.     

Spectroscopic analysis of the unfolding of transition metal-ion complexes of human lactoferrin and transferrin.

1. Human lactoferrin and transferrin are capable of binding several transition metal ions [Fe(III), Cu(II), Mn(III), Co(III)] into specific binding sites in the presence of bicarbonate. 2. Increased conformational stability and increased resistance to protein unfolding is observed for these metal-ion complexes compared to the apoprotein form of these proteins. 3. Mn(III)-lactoferrin and transferrin complexes exhibit steeper denaturation transitions than the Co(III) complexes of these proteins suggesting greater cooperativity in the unfolding process. 4. The incorporation of Fe(III) into the specific metal binding sites offers the greatest resistance to thermal unfolding when compared to the other transition metal ions studied. 5. Non-coincidence of unfolding transitions is observed, with fluorescence transition midpoints being lower than those determined by absorbance measurements. 6. Fully denatured proteins in the presence of urea and alkyl ureas exhibit fluorescence wavelength maxima at 355-356 nm indicative of tryptophan exposure upon protein unfolding.     

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

Conformational change in the stator of the bacterial flagellar motor.

MotA and MotB are integral membrane proteins of Escherichia coli that form the stator of the proton-fueled flagellar rotary motor. The motor contains several MotA/MotB complexes, which function independently to conduct protons across the cytoplasmic membrane and couple proton flow to rotation. MotB contains a conserved aspartic acid residue, Asp32, that is critical for rotation. We have proposed that the protons energizing the motor interact with Asp32 of MotB to induce conformational changes in the stator that drive movement of the rotor. To test for conformational changes, we examined the protease susceptibility of MotA in membrane-bound complexes with either wild-type MotB or MotB mutated at residue 32. Small, uncharged replacements of Asp32 in MotB (D32N, D32A, D32G, D32S, or D32C) caused a significant change in the conformation of MotA, as evidenced by a change in the pattern of proteolytic fragments. The conformational change does not require any flagellar proteins besides MotA and MotB, as it was still seen in a strain that expresses no other flagellar genes. It affects a cytoplasmic domain of MotA that contains residues known to interact with the rotor, consistent with a role in the generation of torque. Influences of key residues of MotA on conformation were also examined. Pro173 of MotA, known to be important for rotation, is a significant determinant of conformation: Dominant Pro173 mutations, but not recessive ones, altered the proteolysis pattern of MotA and also prevented the conformational change induced by Asp32 replacements. Arg90 and Glu98, residues of MotA that engage in electrostatic interactions with the rotor, appear not to be strong determinants of conformation of the MotA/MotB complex in membranes. We note sequence similarity between MotA and ExbB, a cytoplasmic-membrane protein that energizes outer-membrane transport in Gram-negative bacteria. ExbB and associated proteins might also employ a mechanism involving proton-driven conformational change.     

Gel to liquid crystalline phase transition promotes a conformational reorganization of Ca2+, Mg2+-ATPase from sarcoplasmic reticulum in dimyristoylphosphatidylcholine reconstituted systems

Sarcoplasmic reticulum Ca2+, Mg2+-ATPase has been reconstituted in membranes highly enriched in dimyristoylphosphatidylcholine. According to electron microscopy data these membranes form vesicles of an average diameter of 1000 +/- 200 A. These reconstituted membranes show hysteretic behavior in some physical-chemical properties, such as light scattering and fluorescence when labeled with iodoacetamidofluorescein and with N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine. Hysteretic behavior in catalytic activity can also be inferred from the kinetic data presented in this paper, because the temperature dependence of the Ca2+, Mg2+-ATPase activity is altered by a mild thermal pretreatment of the samples. Furthermore, it was noticed that the Ca2+-dependent ATPase activity of these complexes, when assayed above the phase transition temperature (Tc) of the lipid matrix, showed a lag phase in the minute time scale range. On the basis of these findings, it is suggested that the gel-to-liquid crystalline phase transition of the lipid is able to shift the conformational equilibrium E----E* of Ca2+, Mg2+-ATPase. The fact that the -SH reactivity against 5,5'-dithio-bis-nitrobenzoic acid of these complexes is also altered by preincubation above Tc for several minutes also supports that lipid melting induces a conformational change in Ca2+, Mg2+-ATPase.     

Binding of C5-dicarboxylic substrate to aspartate aminotransferase: implications for the conformational change at the transaldimination step

The mechanism for the reaction of aspartate aminotransferase with the C4 substrate, l-aspartate, has been well established. The binding of the C4 substrate induces conformational change in the enzyme from the open to the closed form, and the entire reaction proceeds in the closed form of the enzyme. On the contrary, little is known about the reaction with the C5 substrate, l-glutamate. In this study, we analyzed the pH-dependent binding of 2-methyl-l-glutamate to the enzyme and showed that the interaction between the amino group of 2-methyl-l-glutamate and the pyridoxal 5'-phosphate aldimine is weak compared to that between 2-methyl-l-aspartate and the aldimine. The structures of the Michaelis complexes of the enzyme with l-aspartate and l-glutamate were modeled on the basis of the maleate and glutarate complex structures of the enzyme. The result showed that l-glutamate binds to the open form of the enzyme in an extended conformation, and its alpha-amino group points in the opposite direction of the aldimine, while that of l-aspartate is close to the aldimine. These models explain the observations for 2-methyl-l-glutamate and 2-methyl-l-aspartate. The crystal structures of the complexes of aspartate aminotransferase with phosphopyridoxyl derivatives of l-glutamate, d-glutamate, and 2-methyl-l-glutamate were solved as the models for the external aldimine and ketimine complexes of l-glutamate. All the structures were in the closed form, and the two carboxylate groups and the arginine residues binding them are superimposable on the external aldimine complex with 2-methyl-l-aspartate. Taking these facts altogether, it was strongly suggested that the binding of l-glutamate to aspartate aminotransferase to form the Michaelis complex does not induce a conformational change in the enzyme, and that the conformational change to the closed form occurs during the transaldimination step. The hydrophobic residues of the entrance of the active site, including Tyr70, are considered to be important for promoting the transaldimination process and hence the recognition of the C5 substrate.     

Membrane segment organization in the stator complex of the flagellar motor: implications for proton flow and proton-induced conformational change

MotA and MotB are membrane proteins that form the stator of the bacterial flagellar motor. Each motor contains several MotA 4MotB 2 complexes, which function independently to conduct protons across the membrane and couple proton flow to rotation. The mechanism of rotation is not understood in detail but is thought to involve conformational changes in the stator complexes driven by proton association/dissociation at a critical Asp residue of MotB (Asp 32 in the protein of Escherichia coli). MotA has four membrane segments and MotB has one. Previous studies using targeted disulfide cross-linking showed that the membrane segments of the two MotB subunits are together at the center of the complex, surrounded by the TM3 and TM4 segments of the four MotA subunits. Here, the cross-linking studies are extended to TM1 and TM2 of MotA, using Cys residues introduced in several positions in the segments. The observed patterns of disulfide cross-linking indicate that the TM2 segment is positioned between segments TM3 and TM4 of the same subunit, where it could contribute to the proton-channel-forming part of the structure. TM1 is at the interface between TM4 of its own subunit and the TM3 segment of another subunit, where it could stabilize the complex. A structural model based on the cross-linking results shows unobstructed pathways reaching from the periplasm to the Asp 32 residues near the inner ends of the MotB segments. The model indicates a close proximity for certain conserved, functionally important residues. The results are used to develop an explicit model for the proton-induced conformational change in the stator.     

Antibodies against gangliosides and ganglioside complexes in Guillain–Barré syndrome: New aspects of research

Guillain–Barré syndrome (GBS) is an acute autoimmune neuropathy, often preceded by an infection. Serum anti-ganglioside antibodies are frequently elevated in titer. Those antibodies are useful for diagnosis. Some of them also may be directly involved in the pathogenetic mechanisms by binding to the regions where the respective target ganglioside is specifically localized. We have recently found the presence of the antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex) in the acute-phase sera of some GBS patients. In particular, the antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. Some patients with Miller Fisher syndrome also have antibodies against ganglioside complexes including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. The antibodies against ganglioside complexes may therefore directly cause nerve conduction failure and severe disability in GBS. More study is needed to elucidate the roles of the antibodies against ganglioside complexes.