Shutter Region Research Articles

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

Contrasting conformational dynamics of β-sheet A and helix F with implications in neuroserpin inhibition and aggregation

Neuroserpin (NS) is an inhibitory protein of serpin super family, its shutter region variants have high propensity to aggregate leading to pathological disorders like familial encephalopathy with NS inclusion bodies (FENIB). Helix F and β-sheet A of NS participate in the tissue plasminogen activator (tPA) inhibition but the mechanism is not yet completely understood. A microsecond (μs) molecular dynamics simulation of the helix F and strand 3A variants showed predominant fluctuations in the loop connecting the strands of β-sheet A. Therefore to understand the role of helix F and strand 3A of β-sheet A, cysteine was incorporated at the position N182 in stand 3A (N182C) and position W154 (W154C) in the helix F using site-directed mutagenesis. Purified variants were further labeled with Alexa Fluor488 C5 maleimide dye. Temperature dependent study using non-denaturing PAGE showed the formation of large aggregates of helix F variant W154C but not the strand 3A N182C variant. Interestingly tPA inhibition was found to be decreased in the labeled N182C with decreased tPA-complex formation as compared to labeled W154C NS variant. The fluorescence emission intensity of the labeled helix F variant W154C decreased in the presence of an increasing concentration of tPA, whereas an increase in emission intensity was observed in labeled strand 3A variant N182C, indicating more exposure of strand 3A and shielding of helix F. Taken together the data shows that helix F has a predominant role in the aggregation but a minor role in the inhibition mechanism.

Changes in strand 6B and helix B during neuroserpin inhibition: Implication in severity of clinical phenotype

Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.

Analysis of surface cavity in serpin family reveals potential binding sites for chemical chaperone to reduce polymerization

Serpin constitute about 10% of blood protein and are associated with mutations that results in aberrant intermolecular linkages which leads to polymer formation. Studies with short peptides have shown promise in depolymerization of serpins however a reactive center loop based peptide also makes the serpin inactive. A chemical chaperone based approach is a better option in terms of maintaining activity and retarding polymerization but not much is known about its binding and mechanism. Specific target for chemical chaperones and its effectiveness across many serpin is not known. We did an analysis of serpin cavity using CASTp and show that cavities are distributed throughout the molecule where the largest cavities are generally present in areas of major conformational change like shutter region, helix D and helix F. An analysis of different conformational states of serpins showed that this large cavity undergoes increase in size in latent and cleaved states as compared to native state. We targeted serpins with a variety of carbohydrate, methylamine and amino acid based chemical chaperones and selected those that have highest binding energy across different serpins to assess their ability to bind large cavities. The results show that carbohydrate based chemical chaperone like sorbitol, sucrose, arabitol and trehalose and amino acid based chaperones like dopamine, phenylalanine, arginine and glutamic acid are the most effective in binding serpins. Most of these chemical chaperone interacted with residues in the shutter region and the helix D arm at the C-terminal which are part of the largest cavities. We selected the carbohydrate based chemical chaperone with best binding energies and did experimental study under the condition that induce polymerization and show that indeed they were able to retard polymer formation with moderate effect on inhibition rates. However a fluorometric study with native antithrombin showed that chemical chaperone may effect the conformation of the proteins. Our study shows that chemical chaperones have the best binding affinities for the cavities around shutter region and helix D and that a cavity targeting based approach seems to be a better option for retarding polymerization in serpins, but a thorough analysis of its effect on folding, inhibition and cofactor binding is required.

Crystal structure of protein Z–dependent inhibitor complex shows how protein Z functions as a cofactor in the membrane inhibition of factor X

AbstractProtein Z (PZ) binds to PZ-dependent inhibitor (ZPI) and accelerates the inhibition of the coagulation protease, activated factor X (FXa), in the presence of phospholipids and Ca2+. A 2.3Å resolution crystal structure of PZ complexed with ZPI shows that ZPI is a typical serine protease inhibitor and that PZ has a serine protease fold with distorted oxyanion hole and S1 pocket. The 2 molecules bind with fully complementary surfaces spanning over 2400Å2 and involving extensive ionic and hydrophobic interactions. ZPI has an unusual shutter region with a negatively charged residue buried within the hydrophobic core of the molecule. This unique Asp213 is critical in maintaining the balanced metastability required for optimal protease inhibition, especially when PZ is bound, with its replacement with Asn resulting in increased thermal stability, but decreased efficiency of protease inhibition. The structure of ZPI shows negatively and positively charged surfaces on top of the molecule, in keeping with mutagenesis studies in this work indicating exosite interactions with FXa when it docks on top of ZPI. As modeled in this study, the γ-carboxy-glutamic acid-containing domains of PZ and FXa enable them to bind to the same phospholipid surfaces on platelet and other membranes, with optimal proximity for the inhibition of FXa by the complexed ZPI.

Latent S49P Neuroserpin Forms Polymers in the Dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies

The serpinopathies result from conformational transitions in members of the serine proteinase inhibitor superfamily with aberrant tissue deposition or loss of function. They are typified by mutants of neuroserpin that are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. We show here that the S49P mutant of neuroserpin that causes the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) forms a latent species in vitro and in vivo in addition to the formation of polymers. Latent neuroserpin is thermostable and inactive as a proteinase inhibitor, but activity can be restored by refolding. Strikingly, latent S49P neuroserpin is unlike any other latent serine proteinase inhibitor (serpin) in that it spontaneously forms polymers under physiological conditions. These data provide an alternative method for the inactivation of mutant neuroserpin as a proteinase inhibitor in FENIB and demonstrate a second pathway for the formation of intracellular polymers in association with disease.

The High Resolution Crystal Structure of a Native Thermostable Serpin Reveals the Complex Mechanism Underpinning the Stressed to Relaxed Transition

Serpins fold into a native metastable state and utilize a complex conformational change to inhibit target proteases. An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures. However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment. We have determined the 1.8 A x-ray crystal structure of thermopin in the native, inhibitory conformation. A structural comparison with the previously determined 1.5 A structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins. Flexibility in the shutter region and electrostatic interactions at the top of the A beta-sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state. Here we have discussed the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures.

Mutations in the shutter region of antithrombin result in formation of disulfide‐linked dimers and severe venous thrombosis

Missense mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences of mutations affecting antithrombin, the main hemostatic serpin. To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin. We identified two families with significant reduction of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparin-affinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer. We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal antithrombin, supporting inefficiently expressed rather than non-expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide-linked dimer of mutant antithrombin whose properties are consistent with head-to-head insertion of the reactive loop. Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these mutations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated by pregnancy or infection.

Conserved Ser residues, the Shutter Region, and Speciation in Serpin Evolution

The suicide inhibitory mechanism of serine protease inhibitors of the serpin superfamily depends heavily on their structural flexibility, which is controlled in large part by the breach and shutter regions of the central Abeta-sheet. We examined codon usage by the highly conserved residues, Ser-53 and Ser-56, of the shutter region and found a TCN-AGY usage dichotomy for Ser-56 that remarkably is linked to the protostome-deuterostome split. Our results suggest that serpin evolution was driven by phylogenetic speciation and not pressure to fulfill new physiologic functions mitigating against coevolution with the family of serine proteases they inhibit.

Mutations in the shutter region of anti-thrombin result in severe episodic thrombosis and unlike other serpins in formation of disulfide-linked dimers

Mutations in the shutter region of anti-thrombin result in severe episodic thrombosis and unlike other serpins in formation of disulfide-linked dimers

Serpin Polymerization Is Prevented by a Hydrogen Bond Network That Is Centered on His-334 and Stabilized by Glycerol

Polymerization of serpins commonly results from mutations in the shutter region underlying the bifurcation of strands 3 and 5 of the A-sheet, with entry beyond this point being barred by a H-bond network centered on His-334. Exposure of this histidine in antithrombin, which has a partially opened sheet, allows polymerization and peptide insertion to occur at pH 6 or less when His-334 will be predictably protonated with disruption of the H-bond network. Similarly, thermal stability of antithrombin is pH-dependent with a single unfolding transition at pH 6, but there is no such transition when His-334 is buried by a fully closed A-sheet in heparin-complexed antithrombin or in alpha(1)-antitrypsin. Replacement of His-334 in alpha(1)-antitrypsin by a serine or alanine at pH 7.4 results in the same polymerization and loop-peptide acceptance observed with antithrombin at low pH. The critical role of His-334 and the re-formation of its H-bond network by the conserved P8 threonine, on the full insertion of strand 4, are relevant for the design of therapeutic blocking agents. This is highlighted here by the crystallographic demonstration that glycerol, which at high concentrations blocks polymerization, can replace the P8 threonine and re-form the disrupted H-bond network with His-334.

Importance of the amino-acid composition of the shutter region of plasminogen activator inhibitor-1 for its transitions to latent and substrate forms.

The serpins are of general protein chemical interest due to their ability to undergo a large conformational change consisting of the insertion of the reactive centre loop (RCL), which becomes strand 4, into the central beta sheet A. To make space for the incoming RCL, the 'shutter region' opens by the beta strands 3A and 5A sliding apart over the underlying alpha helix B. Loop insertion occurs during the formation of complexes of serpins with their target serine proteinases and during latency transition. This type of loop insertion is unique to plasminogen activator inhibitor-1 (PAI-1). We report here that amino-acid substitutions in a buried cluster of three residues forming a hydrogen bonding network in the shutter region drastically accelerate PAI-1 latency transition; that the rate was in all cases normalized by the PAI-1 binding protein vitronectin; and that substitution of an adjacent beta strand 5A Lys residue, believed to anchor beta strand 5A to other secondary structural elements, had differential effects on the rates of latency transition in the absence and the presence of vitronectin, respectively. An overlapping, but not identical set of substitutions resulted in an increased tendency to substrate behaviour of PAI-1 at reaction with its target proteinases. These findings show that vitronectin regulates the movements of the RCL through conformational changes of the shutter region and beta strand 5A, are in agreement with RCL insertion proceeding by different routes during latency transition and complex formation, and contribute to the biochemical basis for the potential use of PAI-1 as a therapeutic target in cancer and cardiovascular diseases.