Coarse‐grained and all‐atom modeling of structural states and transitions in hemoglobin

Hemoglobin (Hb), an oxygen‐binding protein composed of four subunits (α1, α2, β1, and β2), is a well‐known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse‐grained (CG) modeling, all‐atom simulation, and structural data from X‐ray crystallography and wide‐angle X‐ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot‐spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all‐atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all‐atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Large‐scale analysis of the dynamics of enzymes

Protein enzymes enable the cell to execute chemical reactions in short time by accelerating the rate of the reactions in a selective manner. The motions or dynamics of the enzymes are essential for their function. Comparison of the dynamics of a set of 1247 nonhomologous enzymes was performed. For each enzyme, the slowest modes of motion are calculated using the Gaussian network model (GNM) and they are globally aligned. Alignment is done using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. Only 96 pairs of proteins were identified to have three similar GNM slow modes with 63 of them having a similar structure. The most frequent slowest mode of motion describes a two domains anticorrelated motion that characterizes at least 23% of the enzymes. Therefore, dynamics uniqueness cannot be accounted for by the slowest mode itself but rather by the combination of several slow modes. Different quaternary structure packing can restrain the motion of enzyme subunits differently and may serve as another mechanism that increases the dynamics uniqueness. Proteins 2013; 81:1910–1918. © 2013 Wiley Periodicals, Inc.     

Dynamical differences of hemoglobin and the ionotropic glutamate receptor in different states revealed by a new dynamics alignment method

A new algorithm for comparison of protein dynamics is presented. Compared protein structures are superposed and their modes of motions are calculated using the anisotropic network model. The obtained modes are aligned using the dynamic programming algorithm of Needleman and Wunsch, commonly used for sequence alignment. Dynamical comparison of hemoglobin in the T and R2 states reveals that the dynamics of the allosteric effector 2,3‐bisphosphoglycerate binding site is different in the two states. These differences can contribute to the selectivity of the effector to the T state. Similar comparison of the ionotropic glutamate receptor in the kainate+(R,R)‐2b and ZK bound states reveals that the kainate+(R,R)‐2b bound states slow modes describe upward motions of ligand binding domain and the transmembrane domain regions. Such motions may lead to the opening of the receptor. The upper lobes of the LBDs of the ZK bound state have a smaller interface with the amino terminal domains above them and have a better ability to move together. The present study exemplifies the use of dynamics comparison as a tool to study protein function. Proteins 2017; 85:1507–1517. © 2014 Wiley Periodicals, Inc.     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

在中国壮族家系中发现的一例-α~T/-α~Q复合β~0β~0珠蛋白基因型

本文报道在我国广西隆林壮族中发现一个罕見的HbQ复合α,β地中海贫血家系。先证者女,18岁,贫血面容,肝脾肿大。化学结构分析确证本Hb变异体为HbQ Thailand[α74(EF3)Asp→His]。血红蛋白组成以及α和β珠蛋白基因分析结果表明,先证者的珠蛋白基因型为-α~Q/-α~T复合β°/β°(IVSI-1G→T/Codon17A→T);先证者父的基因型为-‘α~Q/-复合β~O/β~A(IVSI-1G→T/β~A);先证母的基因型为-α~T/αα复合β~O/β~A(Codon17A→T/β~A)。     

Cooperative fluctuations of unliganded and substrate-bound HIV-1 protease: a structure-based analysis on a variety of conformations from crystallography and molecular dynamics simulations

The dynamics of HIV-1 protease, both in unliganded and substrate-bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV-1 protease are conserved throughout the MD simulations. The flaps and 40's loop of the unliganded HIV-1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate-bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV-1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool.     

Dynamics and allostery of the ionotropic glutamate receptors and the ligand binding domain

The dynamics of the ligand‐binding domain (LBD) and the intact ionotropic glutamate receptor (iGluR) were studied using Gaussian Network Model (GNM) analysis. The dynamics of LBDs with various allosteric modulators is compared using a novel method of multiple alignment of GNM modes of motion. The analysis reveals that allosteric effectors change the dynamics of amino acids at the upper lobe interface of the LBD dimer as well as at the hinge region between the upper‐ and lower‐ lobes. For the intact glutamate receptor the analysis show that the clamshell‐like movement of the LBD upper and lower lobes is coupled to the bending of the trans‐membrane domain (TMD) helices which may open the channel pore. The results offer a new insight on the mechanism of action of allosteric modulators on the iGluR and support the notion of TMD helices bending as a possible mechanism for channel opening. In addition, the study validates the methodology of multiple GNM modes alignment as a useful tool to study allosteric effect and its relation to proteins dynamics. Proteins 2016; 84:267–277. © 2015 Wiley Periodicals, Inc.     

Correspondences between low-energy modes in enzymes: dynamics-based alignment of enzymatic functional families

Proteins that show similarity in their equilibrium dynamics can be aligned by identifying regions that undergo similar concerted movements. These movements are computed from protein native structures using coarse-grained elastic network models. We show the existence of common large-scale movements in enzymes selected from the main functional and structural classes. Alignment via dynamics does not require prior detection of sequence or structural correspondence. Indeed, a third of the statistically significant dynamics-based alignments involve enzymes that lack substantial global or local structural similarities. The analysis of specific residue-residue correspondences of these structurally dissimilar enzymes in some cases suggests a functional relationship of the detected common dynamic features. Including dynamics-based criteria in protein alignment thus provides a promising avenue for relating and grouping enzymes in terms of dynamic aspects that often, though not always, assist or accompany biological function.     

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.     

Structure alignment via Delaunay tetrahedralization

A novel protein structure alignment technique has been developed reducing much of the secondary and tertiary structure to a sequential representation greatly accelerating many structural computations, including alignment. Constructed from incidence relations in the Delaunay tetrahedralization, alignments of the sequential representation describe structural similarities that cannot be expressed with rigid-body superposition and complement existing techniques minimizing root-mean-squared distance through superposition. Restricting to the largest substructure superimposable by a single rigid-body transformation determines an alignment suitable for root-mean-squared distance comparisons and visualization. Restricted alignments of a test set of histones and histone-like proteins determined superpositions nearly identical to those produced by the established structure alignment routines of DaliLite and ProSup. Alignment of three, increasingly complex proteins: ferredoxin, cytidine deaminase, and carbamoyl phosphate synthetase, to themselves, demonstrated previously identified regions of self-similarity. All-against-all similarity index comparisons performed on a test set of 45 class I and class II aminoacyl-tRNA synthetases closely reproduced the results of established distance matrix methods while requiring 1/16 the time. Principal component analysis of pairwise tetrahedral decomposition similarity of 2300 molecular dynamics snapshots of tryptophanyl-tRNA synthetase revealed discrete microstates within the trajectory consistent with experimental results. The method produces results with sufficient efficiency for large-scale multiple structure alignment and is well suited to genomic and evolutionary investigations where no geometric model of similarity is known a priori.     

基于残基相互作用网络比对的木聚糖酶热稳定性的研究

研究木聚糖酶的耐热机理能够提升工业生产效率和经济效益。本文使用提出的SI-MAGNA残基相互作用网络比对算法对来自嗜热子囊菌（Thermoascus aurantiacus）的耐热型木聚糖酶和来自变铅青链霉菌（Streptomyces lividans）的常温型木聚糖酶进行网络比对,以探究影响两者结构稳定性和热稳定性的因素。通过比对结果分析：证实了(βα)8-桶结构的低序列保守性及β折叠对结构稳定性的作用;发现了1E0W的loop1区域中独有一个310-螺旋结构影响了结构稳定性;1TUX中的α1’短螺旋结构和相对较短的β4α4-loop区域有助于提升其结构稳定性和酶的热稳定性;推测1TUX的β4α4-loop区域中独有的氢键转折结构和1E0W的loop6区域中独有的2个β桥结构可能引起空间结构的细微差异,从而影响酶的热稳定性。     

Functional and structural interaction of (−)-lobeline with human α4β2 and α4β4 nicotinic acetylcholine receptor subtypes

To determine the pharmacologic activity of (−)-lobeline between human (h)α4β2 and hα4β4 nicotinic acetylcholine receptors (AChRs), functional and structural experiments were performed. The Ca²⁺ influx results established that (−)-lobeline neither actives nor enhances the function of the studied AChR subtypes, but competitively inhibits hα4β4 AChRs with potency ∼10-fold higher than that for hα4β2 AChRs. This difference is due to a higher binding affinity for the [³H]cytisine sites at hα4β4 compared to hα4β2 AChRs, which, in turn, can be explained by our molecular dynamics (MD) results: (1) higher stability of (−)-lobeline and its hydrogen bonds within the α4β4 pocket compared to the α4β2 pocket, (2) (−)-lobeline promotes Loop C to cap the binding site at the α4β4 pocket, but forces Loop C to get apart from the α4β2 pocket, precluding the gating process elicited by agonists, and (3) the orientation of (−)-lobeline within the α4β4, but not the α4β2, subpocket, promoted by the t− (or t+) rotameric state of α4-Tyr98, remains unchanged during the whole MD simulation. This study gives a detailed view of the molecular and dynamics events evoked by (−)-lobeline supporting the differential binding affinity and subsequent inhibitory potency between hα4β2 and hα4β4 AChRs, and supports the possibility that the latter subtype is also involved in its activity.     

Method for comparing the structures of protein ligand-binding sites and application for predicting protein-drug interactions

Many drugs, even ones that are designed to act selectively on a target protein, bind unintended proteins. These unintended bindings can explain side effects or indicate additional mechanisms for a drug's medicinal properties. Structural similarity between binding sites is one of the reasons for binding to multiple targets. We developed a method for the structural alignment of atoms in the solvent-accessible surface of proteins that uses similarities in the local atomic environment, and carried out all-against-all structural comparisons for 48,347 potential ligand-binding regions from a nonredundant protein structure subset (nrPDB, provided by NCBI). The relationships between the similarity of ligand-binding regions and the similarity of the global structures of the proteins containing the binding regions were examined. We found 10,403 known ligand-binding region pairs whose structures were similar despite having different global folds. Of these, we detected 281 region pairs that had similar ligands with similar binding modes. These proteins are good examples of convergent evolution. In addition, we found a significant correlation between Z-score of structural similarity and true positive rate of "active" entries in the PubChem BioAssay database. Moreover, we confirmed the interaction between ibuprofen and a new target, porcine pancreatic elastase, by NMR experiment. Finally, we used this method to predict new drug-target protein interactions. We obtained 540 predictions for 105 drugs (e.g., captopril, lovastatin, flurbiprofen, metyrapone, and salicylic acid), and calculated the binding affinities using AutoDock simulation. The results of these structural comparisons are available at http://www.tsurumi.yokohama-cu.ac.jp/fold/database.html.     

A biochemical--biophysical study of hemoglobins from woolly mammoth, Asian elephant, and humans

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(β/δ)(1) and α(1)(β/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.     

Structural studies on a low oxygen affinity hemoglobin from mammalian species: Sheep (Ovis aries)

Hemoglobin (Hb) is in equilibrium between low affinity Tense (T) and high affinity Relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express ‘high’ and ‘low’ oxygen affinity Hbs. Although Hbs from former group have been studied extensively, a limited number of structural studies have been performed for the low oxygen affinity Hbs. Here, the crystal structure of low oxygen affinity sheep methemoglobin (metHb) has been determined to 2.7 Å resolution. Even though sheep metHb adopts classical R state like quaternary structure, it shows localized quaternary and tertiary structural differences compared with other liganded Hb. The critical group of residues in the “joint region”, shown as a major source of quaternary constraint on deoxyHb, formed unique interactions in the α1β2/α2β1 interfaces of sheep metHb structure. In addition, the constrained β subunits heme environment and the contraction of N-termini and A-helices of β subunits towards the molecular dyad are observed for sheep metHb structure. These observations provide the structural basis for a low oxygen affinity and blunt response to allosteric effector of sheep Hb.     

Effects of Peutz-Jeghers syndrome (PJS) causing missense mutations L67P,L182P,G242V and R297S on the structural dynamics of LKB1 (Liver kinase B1) protein

The liver kinase B1 (LKB1) is encoded by LKB1 gene. Several pathogenic mutations of LKB1 causing Peutz–Jeghers syndrome and also cancers in breast, gastric, pancreas, and colon have been reported. The present study is focused to analyze the effects on the structural dynamics of LKB1 caused by the 4 pathogenic missense mutations (L67P, L182P, G242V, and R297S), which are reported to reduce the catalytic activity. In this study, the structural changes of LKB1 in apo- and in heterotrimeric complex (LKB1–STRADα–MO25α) form with wild and mutated LKB1 are investigated using all atomistic molecular dynamic simulation. The present study reveals that these four mutations initiate local structural distortions and the solvent accessibility of the surrounding regions of ATP-binding pocket such as glycine-rich loop, αB and αC loop, activation and catalytic loops. The mutations of L67P, L182P, and G242 V induce distortions of the secondary structure of β1–β3 sheets, π – π interaction (observed between Phe204 of LKB1 and Phe243 of MO25α), and increase the helical properties (both helical twist and length) of the adjacent αH-helix, respectively. The active kinase features like the conformation of catalytic and activation loops, salt bridge and, finally, the formation of stable R- and C-hydrophobic spines are also found to be perturbed by these mutations. Hence, the observed mutation-induced structural distortions fail to coordinate the essential binding nature of LKB1 with STRADα and MO25α, which eventually affects the native function of LKB1. These observations are in line with the experimentally reported reduced kinase activity of LKB1.     

Escherichia coli adenylate kinase dynamics: comparison of elastic network model modes with mode-coupling (15)N-NMR relaxation data

The dynamics of adenylate kinase of Escherichia coli (AKeco) and its complex with the inhibitor AP(5)A, are characterized by correlating the theoretical results obtained with the Gaussian Network Model (GNM) and the anisotropic network model (ANM) with the order parameters and correlation times obtained with Slowly Relaxing Local Structure (SRLS) analysis of (15)N-NMR relaxation data. The AMPbd and LID domains of AKeco execute in solution large amplitude motions associated with the catalytic reaction Mg(+2)*ATP + AMP --> Mg(+2)*ADP + ADP. Two sets of correlation times and order parameters were determined by NMR/SRLS for AKeco, attributed to slow (nanoseconds) motions with correlation time tau( perpendicular) and low order parameters, and fast (picoseconds) motions with correlation time tau( parallel) and high order parameters. The structural connotation of these patterns is examined herein by subjecting AKeco and AKeco*AP(5)A to GNM analysis, which yields the dynamic spectrum in terms of slow and fast modes. The low/high NMR order parameters correlate with the slow/fast modes of the backbone elucidated with GNM. Likewise, tau( parallel) and tau( perpendicular) are associated with fast and slow GNM modes, respectively. Catalysis-related domain motion of AMPbd and LID in AKeco, occurring per NMR with correlation time tau( perpendicular), is associated with the first and second collective slow (global) GNM modes. The ANM-predicted deformations of the unliganded enzyme conform to the functional reconfiguration induced by ligand-binding, indicating the structural disposition (or potential) of the enzyme to bind its substrates. It is shown that NMR/SRLS and GNM/ANM analyses can be advantageously synthesized to provide insights into the molecular mechanisms that control biological function.     

Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.     

Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels.

An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure-based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root. The algorithm encoded in STAMP (STructural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases. In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij' provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij' values and thus provide a reproducible resource for studies of residue conservation within structural motifs.     

Insights into the structural dynamics of Liver kinase B1 (LKB1) by the binding of STe20 Related Adapterα (STRADα) and Mouse protein 25α (MO25α) co-activators

LKB1, the tumour suppressor, is found mutated in Peutz-Jeghers syndrome (PJS). The LKB1 is a serine-threonine kinase protein that is allosterically activated by the binding of STRADα and MO25α without phosphorylating the Thr212 present at activation loop. The present study aims to highlight the structural dynamics and complexation mechanism during the allosteric activation of LKB1 by these co-activators using molecular dynamics simulations. The all atom simulations performed on the complexes of LKB1 with ATP, STRADα, and MO25α for a period of 30 ns reveal that binding of STRADα and MO25α significantly stabilizes the highly flexible regions of LKB1 such as ATP binding region (β1-β2 loop), catalytic & activation loop segments and αG helix. Also, binding of STRADα and MO25α to LKB1 promotes coordinated motion between N- and C-lobes along with the catalytic & activation loops by forming H-bonds between LKB1 and co-activators, which further facilitate to establish the conserved attributes of active LKB1 such as (i) formation of salt bridge between Lys78 and Glu98, (ii) formation of stable hydrophobic R- and C-spines, and (iii) interaction between both catalytic and activation loops. Especially, the residues of LKB1 interacting with STRADα (Arg74, Glu342) and MO25α (Glu165, Pro203 and Phe204) are observed to play a significant role in stabilizing the (LKB1-ATP)-(STRADα-ATP)-MO25α complex. Overall, the present work highlighting the structural dynamics of LKB1 by the binding of allosteric co-activators is expected to provide a basic understanding on drug design specific to PJS syndrome.