Flexible Surface Loop Research Articles

A flexible RecC surface loop required for Chi hotspot control of RecBCD enzyme.

Escherichia coli RecBCD helicase-nuclease promotes vital homologous recombination-based repair of DNA double-strand breaks. The RecB nuclease domain (Nuc) is connected to the RecB helicase domain by a 19-amino-acid tether. When DNA binds to RecBCD, published evidence suggests that Nuc moves ∼50 Å from the exit of a RecC tunnel, from which the 3'-ended strand emerges during unwinding, to a distant position on RecC's surface. During subsequent ATP-dependent unwinding of DNA, Nuc nicks the 3'-ended strand near 5'-GCTGGTGG-3' (Chi recombination hotspot). Here, we test our model of Nuc swinging on the tether from the RecC tunnel exit to the RecC distant surface and back to the RecC tunnel exit to cut at Chi. We identify positions in a flexible surface loop on RecC and on RecB Nuc with complementary charges, mutation of which strongly reduces but does not eliminate Chi hotspot activity in cells. The recC loop mutation interacts with recB mutations hypothesized to be in the Chi-activated intramolecular signal transduction pathway; the double mutants, but not the single mutants, eliminate Chi hotspot activity. A RecC amino acid near the flexible loop is also essential for full Chi activity; its alteration likewise synergizes with a signal transduction mutation to eliminate Chi activity. We infer that altering the RecC surface loop reduces coordination among the subunits, which is critical for Chi hotspot activity. We discuss other RecBCD mutants with related properties.

Recognition of a Flexible Protein Loop in Taspase 1 by Multivalent Supramolecular Tweezers.

Many natural proteins contain flexible loops utilizing well-defined complementary surface regions of their interacting partners and usually undergo major structural rearrangements to allow perfect binding. The molecular recognition of such flexible structures is still highly challenging due to the inherent conformational dynamics. Notably, protein-protein interactions are on the other hand characterized by a multivalent display of complementary binding partners to enhance molecular affinity and specificity. Imitating this natural concept, we here report the rational design of advanced multivalent supramolecular tweezers that allow addressing two lysine and arginine clusters on a flexible protein surface loop. The protease Taspase 1, which is involved in cancer development, carries a basic bipartite nuclear localization signal (NLS) and thus interacts with Importin α, a prerequisite for proteolytic activation. Newly established synthesis routes enabled us to covalently fuse several tweezer molecules into multivalent NLS ligands. The resulting bi- up to pentavalent constructs were then systematically compared in comprehensive biochemical assays. In this series, the stepwise increase in valency was robustly reflected by the ligands' gradually enhanced potency to disrupt the interaction of Taspase 1 with Importin α, correlated with both higher binding affinity and inhibition of proteolytic activity.

Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: insights into plasticity of substrate binding and activation.

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomyces antibioticus termed TNYR SaPLD was developed capable of synthesising 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild-type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry

Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O2-reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Å from the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme.

Enhancing thermostability by modifying flexible surface loops in an evolved high‐redox potential laccase

AbstractHigh‐redox potential laccases (HRPLs) from white‐rot fungi are versatile biocatalysts whose practical use is highly dependent on their thermostability. In this work, an evolved HRPL variant was subjected to structure‐guided evolution to improve its thermostability. We first selected several surface flexible loops in the laccase structure by inspecting them through molecular dynamics and an analysis of B‐factors. The resulting segments were grouped into three MORPHING (Mutagenic Organized Recombination Process by Homologous In vivo Grouping) blocks, which were constructed in Saccharomyces cerevisiae and explored at high temperatures. This evolution process gave rise to a double mutant that showed a half‐life at 70°C enhanced by 31 min with an optimum temperature for activity of 75°C and similar kinetic parameters. The Ser264Lys and Ser356Asn mutations modified the contacts established between these residues and those that surround them, altering the surface loops and thereby the enzyme properties.

Crystal structure and substrate interactions of an unusual fungal non-CBM carrying GH26 endo-\u03b2-mannanase from Yunnania penicillata

Endo-β(1 → 4)-mannanases (endomannanases) catalyse degradation of β-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the −3 and −2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the −2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation.

Improvement in the thermostability of chitosanase from Bacillus ehimensis by introducing artificial disulfide bonds.

To determine the effects of artificial disulfide bridges on the thermostability and catalytic efficiency of chitosanase EAG1. Five artificial disulfide bridges were designed based on the structural information derived from the three-dimensional (3-D) model of chitosanase EAG1. Two beneficial mutants (G113C/D116C, A207C-L286C) were located in the flexible surface loop region, whereas the similar substitutions introduced in α-helices regions had a negligible effect. Mut5, the most active mutant, had a longer half-life at 50°C (from 10.5 to 69.3min) and a 200% higher catalytic efficiency (K cat/K m) than that of the original EAG1. The contribution of disulfide bridges to enzyme thermostability is mainly dependent on its location within the polypeptide chain. Strategical placement of a disulfide bridge in flexible regions provides a rigid support and creation of a protected microenvironment, which is effective in improving enzyme's thermostability and catalytic efficiency.

A new crystal form of a hyperthermophilic endocellulase.

The hyperthermophilic glycoside hydrolase family endocellulase 12 from the archaeon Pyrococcus furiosus (EGPf; Gene ID PF0854; EC 3.2.1.4) catalyzes the hydrolytic cleavage of the β-1,4-glucosidic linkage in β-glucan in lignocellulose biomass. A crystal of EGPf was previously prepared at pH 9.0 and its structure was determined at an atomic resolution of 1.07 Å. This article reports the crystallization of EGPf at the more physiologically relevant pH of 5.5. Structure determination showed that this new crystal form has the symmetry of space group C2. Two molecules of the enzyme are observed in the asymmetric unit. Crystal packing is weak at pH 5.5 owing to two flexible interfaces between symmetry-related molecules. Comparison of the EGPf structures obtained at pH 9.0 and pH 5.5 reveals a significant conformational difference at the active centre and in the surface loops. The interfaces in the vicinity of the flexible surface loops impact the quality of the EGPf crystal.

Structure of the Homodimeric Glycine Decarboxylase P-protein from Synechocystis sp. PCC 6803 Suggests a Mechanism for Redox Regulation

Glycine decarboxylase, or P-protein, is a pyridoxal 5'-phosphate (PLP)-dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an α2 homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apoenzyme shows the C terminus locked in a closed conformation by a disulfide bond between Cys(972) in the C terminus and Cys(353) located in the active site. The presence of the disulfide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys(972) and Cys(353) by Ser using site-directed mutagenesis have distinctly lower specific activities, supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353-972 disulfide releases the C terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulfide bond that acts to stabilize the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier.

First Three-Dimensional Structure of Toxoplasma gondii Thymidylate Synthase–Dihydrofolate Reductase: Insights for Catalysis, Interdomain Interactions, and Substrate Channeling

Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Toxoplasma gondii , contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the catalytic activities contained on a single polypeptide chain. The prevalence of T. gondii infections across the world, especially for those immunocompromised, underscores the need to understand TS-DHFR enzyme function and to find new avenues to exploit for the design of novel antiparasitic drugs. As a first step, we have solved the first three-dimensional structures of T. gondii TS-DHFR at 3.7 Å and of a loop truncated TS-DHFR, removing several flexible surface loops in the DHFR domain, improving resolution to 2.2 Å. Distinct structural features of the TS-DHFR homodimer include a junctional region containing a kinked crossover helix between the DHFR domains of the two adjacent monomers, a long linker connecting the TS and DHFR domains, and a DHFR domain that is positively charged. The roles of these unique structural features were probed by site-directed mutagenesis coupled with presteady state and steady state kinetics. Mutational analysis of the crossover helix region combined with kinetic characterization established the importance of this region not only in DHFR catalysis but also in modulating the distal TS activity, suggesting a role for TS-DHFR interdomain interactions. Additional kinetic studies revealed that substrate channeling occurs in which dihydrofolate is directly transferred from the TS to DHFR active site without entering bulk solution. The crystal structure suggests that the positively charged DHFR domain governs this electrostatically mediated movement of dihydrofolate, preventing release from the enzyme. Taken together, these structural and kinetic studies reveal unique, functional regions on the T. gondii TS-DHFR enzyme that may be targeted for inhibition, thus paving the way for designing species specific inhibitors.

The Sequence and Function Relationship in Ligand-Induced Folding

The Escherichia coli biotin repressor, BirA, the central protein of the E. coli Biotin Regulatory System serves as a metabolic enzyme and the transcription repressor of the biotin biosynthetic operon. For both functions, the active BirA species is bound to bio-5'-AMP, which it synthesizes from substrates biotin and ATP. As an enzyme, BirA catalyzes post-translational biotin linkage to a specific lysine residue of the biotin carboxyl carrier protein (BCCP), a subunit of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis. Transcription repression requires self-association of the adenylate-bound BirA followed by site-specific DNA binding to the biotin operator of the biotin biosynthetic operon. Both functions require folding of two flexible surface loops including the biotin-binding loop (BBL), comprised of residues 116-124, that folds over the biotin moiety and the adenylate-binding loop (ABL), comprised of residues 212-234, that folds over the adenine moiety of the adenylate. In the BirA: adenylate structure side chains of residues F124 and P126 of the BBL and M211 and V218 of the ABL bridge the two loops. Combined kinetic and equilibrium measurements of the consequences of single alanine substitutions of these bridging residues indicate heterogeneity in the effects on the multiple BirA functions. Isothermal titration calorimetry and stopped-flow fluorescence measurements reveal that the substitutions significantly compromise both bio-5'-AMP binding and synthesis. By contrast, these same substitutions have modest effects on homodimerization and biotin transfer to BCCP. These results in combination with previous studies of amino acid replacements in three ABL residues that form a hydrophobic cluster concomitant with adenylate binding indicate a ligand-linked folding process that is characterized by a complex relationship between sequence and function.

Loop 1 dynamics in smooth muscle myosin: isoform specific differences modulate ADP release

Isoforms of the smooth muscle (SM) myosin motor domain differ in the presence or absence of a seven amino acid insert in a flexible surface loop spanning the nucleotide-binding pocket known as Loop 1. The presence of this insert leads to a two-fold increase in actin sliding velocity and ADP release rate between these isoforms, although the effect of Loop 1 on the kinetics of ADP release remains unclear. To further investigate the role of the Loop 1 insert in modulating ADP release in SM myosin we have inserted a single tryptophan residue into Loop 1 of both isoforms as a probe of local structural dynamics. By monitoring the dynamics of Loop 1 in relation to the release of ADP we have observed a unique movement of Loop 1 in the inserted isoform, preceding nucleotide release, which is absent in the non-inserted isoform. This movement is sequence dependent as alanine replacement of the insert residues abolishes the transition and slows ADP release. Thus movement of Loop 1 is a critical factor in increasing the ADP release rate in the inserted faster isoform of SM myosin.

Phospholipid Binding and Membrane Attachment of the Osh4 Protein

Osh4 is an oxysterol binding protein (OSBP) homologue found in yeast that is essential for the intracellular transport of sterols and for cell life. It has been proposed that Osh4 acts as a lipid transport protein, capable of binding a single sterol residue within a hydrophobic binding pocket and transporting it, against a concentration gradient, from the endoplasmic reticulum to the plasma membrane. Phosphoinositides (PIPs) are thought to stimulate sterol transfer by binding to the Osh4 protein surface. In order to study how the Osh4 protein attaches to the plasma membrane, possible lipid binding sites were investigated through the use of blind docking techniques. Model ligand compounds for phosphatidylcholine, phosphatidylserine, and two PIP [PI(4,5)P2 and PI(3,4,5)P3] head groups were docked against several conformational snapshots of the Osh4 surface to determine possible regions favorable to interact with plasma membrane lipids. These conformational snapshots were taken from two 25-ns molecular dynamics simulations of the Osh4 protein complexed with ergosterol and two complexed with 25-hydroxycholesterol. The PIP models frequently docked to a lysine-rich region on an exposed portion of the protein's β-barrel. This region is bounded by a flexible surface loop that is believed to be important for Osh4-membrane binding. Osh4-membrane interaction was also investigated through molecular dynamics simulations of a combined membrane and protein system. Model sterol-acceptor and sterol-donor membranes were constructed using CHARMM-GUI and equilibrated for 25 ns. Key residues of the Osh4 protein that were identified during the blind docking tests, as well as residues known to be in close contact with these membranes upon binding, were placed parallel to the membrane surface. Ultimately, understanding how Osh4 attaches to cellular membranes will lead to a clear understanding how this protein transports sterols in vivo.

Solution Structure and Activation Mechanism of Ubiquitin-Like Small Archaeal Modifier Proteins

In archaea, two ubiquitin-like small archaeal modifier protein (SAMPs) were recently shown to be conjugated to proteins in vivo. SAMPs display homology to bacterial MoaD sulfur transfer proteins and eukaryotic ubiquitin-like proteins, and they share with them the conserved C-terminal glycine–glycine motif. Here, we report the solution structure of SAMP1 from Methanosarcina acetivorans and the activation of SAMPs by an archaeal protein with homology to eukaryotic E1 enzymes. Our results show that SAMP1 possesses a β-grasp fold and that its hydrophobic and electrostatic surface features are similar to those of MoaD. M. acetivorans SAMP1 exhibits an extensive flexible surface loop between helix-2 and the third strand of the β-sheet, which contributes to an elongated surface groove that is not observed in bacterial ubiquitin homologues and many other SAMPs. We provide in vitro biochemical evidence that SAMPs are activated in an ATP-dependent manner by an E1-like enzyme that we have termed E1-like SAMP activator (ELSA). We show that activation occurs by formation of a mixed anhydride (adenylate) at the SAMP C-terminus and is detectable by SDS-PAGE and electrospray ionization mass spectrometry.

A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal: II. A MODEL IN WHICH PHOSPHORYLATION AT SER269 INDUCES A MUTANT CONFORMATION TO p53

The p53 DNA-binding domain harbors a conformationally flexible multiprotein binding site that regulates p53 ubiquitination. A novel phosphorylation site exists within this region at Ser(269), whose phosphomimetic mutation inactivates p53. The phosphomimetic p53 (S269D) exhibits characteristics of mutant p53: stable binding to Hsp70 in vivo, elevated ubiquitination in vivo, inactivity in DNA binding and transcription, increased thermoinstability using thermal shift assays, and λ(max) of intrinsic tryptophan fluorescence at 403 nm rather than 346 nm, characteristic of wild type p53. These data indicate that p53 conformational stability is regulated by a phosphoacceptor site within an exposed flexible surface loop and that this can be destabilized by phosphorylation. To test whether other motifs within p53 have similarly evolved, we analyzed the effect of Ser(215) mutation on p53 function because Ser(215) is another inactivating phosphorylation site in the conformationally flexible PAb240 epitope. The p53(S215D) protein is inactive like p53(S269D), whereas p53(S215A) is as active as p53(S269A). However, the double mutant p53(S215A/S269A) was transcriptionally inactive and more thermally unstable than either individual Ser-Ala loop mutant. Molecular dynamics simulations suggest that (i) solvation of phospho-Ser(215) and phospho-Ser(269) by positive charged residues or solvent water leads to local unfolding, which is accompanied by local destabilization of the N-terminal loop and global destabilization of p53, and (ii) the double alanine 215/269 mutation disrupts hydrogen bonding normally stabilized by both Ser(215) and Ser(269). These data indicate that p53 has evolved two serine phosphoacceptor residues within conformationally flexible epitopes that normally stabilize the p53 DNA-binding domain but whose phosphorylation induces a mutant conformation to wild type p53.

Motif‐based search for a novel fructosyl peptide oxidase from genome databases

The measurement of glycated hemoglobin A1c (HbA1c) has important implications for diagnosis of diabetes and assessment of treatment effectiveness. We proposed specific sequence motifs to identify enzymes that oxidize glycated compounds from genome database searches. The gene encoding a putative fructosyl amino acid oxidase was found in the Phaeosphaeria nodorum SN15 genome and successfully expressed in Escherichia coli. The recombinant protein (XP_001798711) was confirmed to be a novel fructosyl peptide oxidase (FPOX) with high specificity for alpha-glycated compounds, such as HbA1c model compounds fructosyl-(alpha)N-valine (f-(alpha)Val) and fructosyl-(alpha)N-valyl-histidine (f-(alpha)Val-His). Unlike previously reported FPOXs, the P. nodorum FPOX has a K(m) value for f-(alpha)Val-His (0.185 mM) that is considerably lower than that for f-(alpha)Val (0.458 mM). Based on amino acid sequence alignment, three dimensional structural modeling, and site-directed mutagenesis, Gly60 was found to be a determining residue for the activity towards f-(alpha)Val-His. A flexible surface loop region was also found to likely play an important role in accepting f-(alpha)Val-His.

A Kinetic Step Involving Loop 1 in Smooth Muscle may Dictate Isoform Specific Differences in ADP Release

The actin sliding velocity and ADP release rates in smooth muscle myosin are modulated by the make-up of a flexible surface loop spanning the active site known as loop 1. There are only two motor domain isoforms of smooth muscle myosin and they differ in the presence (SM-B) or absence (SM-A) of a seven amino acid insert (QGPSFSY) in this loop. The presence of this insert leads to a two-fold increase in both actin sliding velocity and ADP release, although the mechanism for this difference is unknown. To investigate the role of this insert in functional differences between the SM-B and SM-A isoforms of smooth muscle myosin we have inserted a single tryptophan residue into loop 1 of both isoforms. The dynamics of loop 1 have been correlated with the kinetics of ADP release using a combination of steady-state fluorescence measurements (i.e., tryptophan emission, FRET, and acrylamide quenching) and stopped-flow kinetics. Using this approach we have already shown that the long loop SM-B isoform displays an extra step in its ADP release pathway that has not been previously observed. Here, we show that the additional transition seen in the long loop SM-B isoform is not observed in the short loop SM-A isoform upon ADP release. Furthermore, the final ADP release step is twice as slow in the short loop SM-A isoform, suggesting that the unique transition observed in the presence of the insert alters loop 1 dynamics in a way as to facilitate ADP release. This alteration of ADP release constitutes a simple and fundamental way to tune the activity of the motor at the molecular level and mechanical function at the physiological level in smooth muscle.

Crystal Structure of the Deglycating Enzyme Fructosamine Oxidase (Amadoriase II)

Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6A resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12A deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.

Stability of the Free and Bound Microstates of a Mobile Loop of α-Amylase Obtained from the Absolute Entropy and Free Energy.

The hypothetical scanning molecular dynamics (HSMD) method is a relatively new technique for calculating the absolute entropy, S, and free energy, F, from a given sample generated by any simulation procedure. Thus, each sample conformation, i, is reconstructed by calculating transition probabilities that their product leads to the probability of i, hence to the entropy. HSMD is an exact method where all interactions are considered, and the only approximation is due to insufficient sampling. In previous studies HSMD (and HS Monte Carlo - HSMC) has been applied very successfully to liquid argon, TIP3P water, self-avoiding walks, and peptides in a α-helix, extended, and hairpin microstates. In this paper HSMD is developed further as applied to the flexible 7-residue surface loop, 304-310 (Gly-His-Gly-Ala-Gly-Gly-Ser) of the enzyme porcine pancreatic α-amylase. We are mainly interested in entropy and free energy differences ΔS = Sfree - Sbound (and ΔF=Ffree-Fbound) between the free and bound microstates of the loop, which are obtained from two separate MD samples of these microstates without the need to carry out thermodynamic integration. As for peptides, we find that relatively large systematic errors in Sfree and Sbound (and Ffree and Fbound) are cancelled in ΔS (ΔF) which is thus obtained efficiently with high accuracy, i.e., with a statistical error of 0.1-0.2 kcal/mol (T=300 K) using the AMBER force field and AMBER with the implicit solvation GB/SA. We provide theoretical arguments in support of this cancellation, discuss in detail the problems involved in the computational definition of a microstate in conformational space, suggest potential ways for enhancing efficiency further, and describe the next development where explicit water will replace implicit solvation.

C1 Inhibitor Serpin Domain Structure Reveals the Likely Mechanism of Heparin Potentiation and Conformational Disease

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.