Catalytic Loop Research Articles

Dynamic Flexibility Index Sheds Light on Pin1 Allostery

Allostery, which is regulation from distant sites, plays a major role in biology. Pin1 is a modular protein containing a WW domain and a larger peptidyl prolyl isomarase domain (PPIase) that isomerizes phospho-serine/threonine-proline (pS/T-P) motifs, which are critical for signaling within intrinsically disordered loops of cell cycle proteins. Pin1 utilizes allosteric regulations for its function, and binds (pS/T-P) motifs in both domains. The WW domain serves as a docking module, whereas catalysis solely takes place within the PPIase domain. However, enzymatic activity gets enhanced when WW is in the bound form, highlighting PIN1's allosteric regulation. Previous work using NMR and molecular dynamics analysis has shown that binding induced quenching of fast local motions and strengthening of the interaction between two domains, indicated in particular by decrease in flexibility of catalytic loops. Here we present a novel method, the dynamic flexibility index (DFI) analysis, for characterizing the underlying allosteric communications between two domains. DFI measures the resilience of a given position to the perturbations that occur at different parts of the protein, using linear response theory. This index captures multi-dimensional effects when the protein is displaced out of equilibrium. Moreover, we can also identify the allosteric response in dynamic flexibility based on the perturbation response fluctuation profile of the PPIase domain upon WW binding and distinguish the positions that contribute the most. Finally we also explore the mechanistic link between conformational dynamics and co-evolution to identify mutational positions to alter enzymatic function.

Thermal activation of \u2018allosteric-like\u2019 large-scale motions in a eukaryotic Lactate Dehydrogenase

Conformational changes occurring during the enzymatic turnover are essential for the regulation of protein functionality. Individuating the protein regions involved in these changes and the associated mechanical modes is still a challenge at both experimental and theoretical levels. We present here a detailed investigation of the thermal activation of the functional modes and conformational changes in a eukaryotic Lactate Dehydrogenase enzyme (LDH). Neutron Spin Echo spectroscopy and Molecular Dynamics simulations were used to uncover the characteristic length- and timescales of the LDH nanoscale motions in the apo state. The modes involving the catalytic loop and the mobile region around the binding site are activated at room temperature, and match the allosteric reorganisation of bacterial LDHs. In a temperature window of about 15 degrees, these modes render the protein flexible enough and capable of reorganising the active site toward reactive configurations. On the other hand an excess of thermal excitation leads to the distortion of the protein matrix with a possible anti-catalytic effect. Thus, the temperature activates eukaryotic LDHs via the same conformational changes observed in the allosteric bacterial LDHs. Our investigation provides an extended molecular picture of eukaryotic LDH’s conformational landscape that enriches the static view based on crystallographic studies alone.

Sclerotiorin inhibits protein kinase G from Mycobacterium tuberculosis and impairs mycobacterial growth in macrophages

As a eukaryotic-like Ser/Thr protein kinase, Mycobacterium tuberculosis virulent effector protein kinase G (PknG) mediates mycobacterial survival by regulating bacterial cell metabolic processes and preventing phagosome-lysosome fusion in host macrophages. Targeting PknG is an effective strategy for development of anti-tuberculosis (TB) drugs. In the study, we found that sclerotiorin, derived from marine fungi from the South China Sea, exhibited moderately strong inhibitory effects on recombinant PknG, with an IC50 value of 76.5 μM, and acted as a non-competitive inhibitor. The dissociation constant (KD) of sclerotiorin determined by MST was 11.4 μM, demonstrating a moderate binding strength between them. Sclerotiorin could substantially impair the mycobacterial survival in infected macrophages while the macrophage viability remained unaffected, though it did not inhibit the mycobacterial growth in culture. When sclerotiorin was used in combination with rifampicin, intracellular mycobacterial growth decreased as sclerotiorin concentration increased. Docking analysis suggested a binding mechanism of inhibition with performing interactions with the P-loop and catalytic loop of PknG. In summary, we reported that sclerotiorin had moderately strong PknG inhibitory activity, but no cytotoxicity, and it could substantially decrease the mycobacterial growth inside macrophages, suggesting that sclerotiorin has potential to supplement antibiotic therapy for TB.

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1.

Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency.

Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (kcat/Km , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (kcat/Km ) of these enzymes (in descending order, NfEG12A [100%], NfEG12A-FN [140%], and NfEG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (kcat). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (kcat). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.

Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance.

In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of S. aureus TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase.

A Network of Conformational Transitions Revealed by Molecular Dynamics Simulations of the Binary Complex of Escherichia coli 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase with MgATP.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthetic pathway. Comparison of its X-ray and nuclear magnetic resonance structures suggests that the enzyme undergoes significant conformational change upon binding to its substrates, especially in three catalytic loops. Experimental research has shown that, in its binary form, even bound by analogues of MgATP, loops 2 and 3 remain rather flexible; this raises questions about the putative large-scale induced-fit conformational change of the HPPK-MgATP binary complex. In this work, long-time all-atomic molecular dynamics simulations were conducted to investigate the loop dynamics in this complex. Our simulations show that, with loop 3 closed, multiple conformations of loop 2, including the open, semiopen, and closed forms, are all accessible to the binary complex. These results provide valuable structural insights into the details of conformational changes upon 6-hydroxymethyl-7,8-dihydropterin (HP) binding and biological activities of HPPK. Conformational network analysis and principal component analysis related to the loops are also discussed.

Computational modeling and molecular dynamics simulations of mammalian cytoplasmic tyrosyl-tRNA synthetase and its complexes with substrates

Cytoplasmic tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. TyrRSs of pathogenic organisms have gained attention as potential targets for drug development. Identifying structural differences between various TyrRSs will facilitate the development of specific inhibitors for the TyrRSs of pathogenic organisms. However, there is a deficiency in structural data for mammalian cytoplasmic TyrRS in complexes with substrates. In this work, we constructed spatial structure of full-length Bos taurus TyrRS (BtTyrRS) and its complexes with substrates using the set of computational modeling techniques. Special attention was paid to BtTyrRS complexes with substrates [L-tyrosine, K+ and ATP:Mg2+] and intermediate products [tyrosyl-adenylate (Tyr-AMP), K+ and PPi:Mg2+] with the different catalytic loop conformations. In order to analyze their dynamical properties, we performed 100 ns of molecular dynamics (MD) simulations. MD simulations revealed new structural data concerning the tyrosine activation reaction in mammalian TyrRS. Formation of strong interaction between Lys154 and γ-phosphate suggests the additional role of CP1 insertion as an important factor for ATP binding. The presence of a potassium-binding pocket within the active site of mammalian TyrRS compensates the absence of the second lysine in the KMSKS motif. Our data provide new details concerning a role of K+ ions at different stages of the first step of the tyrosylation reaction, including the coordination of substrates and involvement in the PPi releasing. The results of this work suggest that differences between ATP-binding sites of mammalian and bacterial TyrRSs are meaningful and could be exploited in the drug design.

The Mechanism of Hsp90 ATPase Stimulation by Aha1.

Hsp90 is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called ‘clients’. Numerous co-chaperone proteins regulate progression through the ATP-dependent client activation cycle. The most potent stimulator of the Hsp90 ATPase activity is the co-chaperone Aha1p. Only one molecule of Aha1p is required to fully stimulate the Hsp90 dimer despite the existence of two, presumably identical, binding sites for this regulator. Using ATPase assays with Hsp90 heterodimers, we find that Aha1p stimulates ATPase activity by a three-step mechanism via the catalytic loop in the middle domain of Hsp90. Binding of the Aha1p N domain to the Hsp90 middle domain exerts a small stimulatory effect but also drives a separate conformational rearrangement in the Hsp90 N domains. This second event drives a rearrangement in the N domain of the opposite subunit and is required for the stimulatory action of the Aha1p C domain. Furthermore, the second event can be blocked by a mutation in one subunit of the Hsp90 dimer but not the other. This work provides a foundation for understanding how post-translational modifications regulate co-chaperone engagement with the Hsp90 dimer.

Autophosphorylation of Specific Threonine and Tyrosine Residues in Arabidopsis CERK1 is Essential for the Activation of Chitin-Induced Immune Signaling.

Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.

Description of Loxtox protein family and identification of a new group of Phospholipases D from Loxosceles similis venom gland

Envenoming resulting from Loxosceles spider bites (loxoscelism) is a recognized public health problem in Brazil. However, the pathophysiology of loxoscelism caused by L. similis bites, which is widespread in Brazil, remains poorly understood. In the present work, the RNA sequencing (RNA-Seq – Next Generation sequencing - NGS) of the L. similis venom gland was performed to identify and analyze the sequences of the key component phospholipase D. The sequences were aligned based on their classical domains, and a phylogenetic tree was constructed. In the bioinformatics analysis, 23 complete sequences of phospholipase D proteins were found and classified as Loxtox proteins, as they contained the characteristic domains of phospholipase D: the active site, the Mg2+-binding domain, and the catalytic loop. Three phospholipase D sequences with non-canonical domains were also found in this work. They were analyzed separately and named PLDs from L. similis (PLD-Ls). This study is the first to characterize phospholipase D sequences from Loxosceles spiders by RNA-Seq. These results contribute new knowledge about the composition of L. similis venom, revealing novel tools that could be used for pharmacological, immunological, and biotechnological applications.

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.

Understanding the role of dynamics in the iron sulfur cluster molecular machine

BackgroundThe bacterial proteins IscS, IscU and CyaY, the bacterial orthologue of frataxin, play an essential role in the biological machine that assembles the prosthetic FeS cluster groups on proteins. They form functionally binary and ternary complexes both in vivo and in vitro. Yet, the mechanism by which they work remains unclear. MethodsWe carried out extensive molecular dynamics simulations to understand the nature of their interactions and the role of dynamics starting from the crystal structure of a IscS-IscU complex and the experimentally-based model of a ternary IscS-IscU-CyaY complex and used nuclear magnetic resonance to experimentally test the interface. ResultsWe show that, while being firmly anchored to IscS, IscU has a pivotal motion around the interface. Our results also describe how the catalytic loop of IscS can flip conformation to allow FeS cluster assembly. This motion is hampered in the ternary complex explaining its inhibitory properties in cluster formation. ConclusionsWe conclude that the observed ‘fluid’ IscS-IscU interface provides the binary complex with a functional adaptability exploited in partner recognition and unravels the molecular determinants of the reported inhibitory action of CyaY in the IscS-IscU-CyaY complex explained in terms of the hampering effect on specific IscU-IscS movements. General significanceOur study provides the first mechanistic basis to explain how the IscS-IscU complex selects its binding partners and supports the inhibitory role of CyaY in the ternary complex.

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.

Molecular Basis for Redox Activation of Epidermal Growth Factor Receptor Kinase

Epidermal growth factor receptor (EGFR) is a target of signal-derived H2O2, and oxidation of active-site cysteine 797 to sulfenic acid enhances kinase activity. Although a major class of covalent drugs targets C797, nothing is known about its catalytic importance or how S-sulfenylation leads to activation. Here, we report the first detailed functional analysis of C797. In contrast to prior assumptions, mutation of C797 diminishes catalytic efficiency invitro and cells. The experimentally determined pKa and reactivity of C797 toward H2O2 correspondingly distinguish this residue from the bulk of the cysteinome. Molecular dynamics simulation of reduced versus oxidized EGFR, reinforced by experimental testing, indicates that sulfenylation of C797 allows new electrostatic interactions to be formed with the catalytic loop. Finally, we show that chronic oxidative stress yields an EGFR subpopulation that is refractory to the FDA-approved drug afatinib. Collectively, our data highlight the significance of redox biology to understanding kinase regulation and drug pharmacology.

Comprehensive structural analysis of the open and closed conformations of Theileria annulata enolase by molecular modelling and docking

Theileria annulata is an apicomplexan parasite which is responsible for tropical theileriosis in cattle. Due to resistance of T. annulata against commonly used antitheilerial drug, new drug candidates should be identified urgently. Enolase might be a druggable protein candidate which has an important role in glycolysis, and could also be related to several cellular functions as a moonlight protein. In this study; we have described three-dimensional models of open and closed conformations of T. annulata enolase by homology modeling method for the first time with the comprehensive domain, active site and docking analyses. Our results show that the enolase has similar folding patterns within enolase superfamily with conserved catalytic loops and active site residues. We have described specific insertions, possible plasminogen binding sites, electrostatic potential surfaces and positively charged pockets as druggable regions in T. annulata enolase.

Somatic mutations in histiocytic sarcoma identified by next generation sequencing.

Histiocytic sarcoma is a rare malignant neoplasm of presumed hematopoietic origin showing morphologic and immunophenotypic evidence of histiocytic differentiation. Somatic mutation importance in the pathogenesis or disease progression of histiocytic sarcoma was largely unknown. To identify somatic mutations in histiocytic sarcoma, we studied 5 histiocytic sarcomas [3 female and 2 male patients; mean age 54.8 (20-72), anatomic sites include lymph node, uterus, and pleura] and matched normal tissues from each patient as germ line controls. Somatic mutations in 50 "Hotspot" oncogenes and tumor suppressor genes were examined using next generation sequencing. Three (out of five) histiocytic sarcoma cases carried somatic mutations in BRAF. Among them, G464V [variant frequency (VF) of 43.6%] and G466R (VF of 29.6%) located at the P loop potentially interfere with the hydrophobic interaction between P and activating loops and ultimately activation of BRAF. Also detected was BRAF somatic mutation N581S (VF of 7.4%), which was located at the catalytic loop of BRAF kinase domain: its role in modifying kinase activity was unclear. A similar mutational analysis was also performed on nine acute monocytic/monoblastic leukemia cases, which did not identify any BRAF somatic mutations. Our study detected several BRAF mutations in histiocytic sarcomas, which may be important in understanding the tumorigenesis of this rare neoplasm and providing mechanisms for potential therapeutical opportunities.

Mechanism of Sulfur Transfer Across Protein–Protein Interfaces: The Cysteine Desulfurase Model System

CsdA cysteine desulfurase (the sulfur donor) and the CsdE sulfur acceptor are involved in biological sulfur trafficking and in iron–sulfur cluster assembly in the model bacterium Escherichia coli. CsdA and CsdE form a stable complex through a polar interface that includes CsdA Cys328 and CsdE Cys61, the two residues known to be involved in the sulfur transfer reaction. Although mechanisms for the transfer of a sulfur moiety across protein–protein interfaces have been proposed based on the IscS–IscU and IscS–TusA structures, the flexibility of the catalytic cysteine loops involved has precluded a high resolution view of the active-site geometry and chemical environment for sulfur transfer. Here, we have used a combination of X-ray crystallography, solution NMR and SAXS, isothermal calorimetry, and computational chemistry methods to unravel how CsdA provides a specific recognition platform for CsdE and how their complex organizes a composite functional reaction environment. The X-ray structures of persulfur...

Evaluating the immunostimulatory characteristics of LicKM, a novel carrier protein.

Abstract The key objective of vaccination is the induction of an effective pathogen-specific immune response that leads to protection against infection and/or disease caused by that pathogen. New vaccine technologies have resulted in second-generation recombinant vaccines containing highly purified antigens with improved tolerability and safety profiles. Unfortunately, the immune responses they induce are suboptimal without the help of adjuvants. Here, utilizing a non-toxic bacterial carrier molecule from Clostridium thermocellum a modified Lichenase (LicKM) and based upon preliminary results we hypothesize, that this serves a dual purpose; as both a mechanism to carrier dominant immunogenic epitopes for presentation to antigen presenting cells during vaccination and as an adjuvant enhancing immunity. This thermostable enzyme contains a catalytic domain loop structure separating it into two regions; an N-terminal and C-terminal region. Target protein sequences are expressed as either N or C terminal fusions; and this molecule, LicKM, may contain a single or double fusion. Currently, we are investigating the mechanism by which this molecule enhances the host humoral and cell-mediated response to vaccination. At this time, we have determined that LicKM physically binds to cells associated with the immune response. Additionally, murine vaccination in the presence of LicKM enhances antibody isotype titer production and possibly enhances the cellular responses. Our goal is to understand the immunostimulatory capability of this protein for its possible use to enhance future vaccination protocols.

Conservation of structural fluctuations in homologous protein kinases and its implications on functional sites.

Our aim is to explore the similarities in structural fluctuations of homologous kinases. Gaussian Network Model based Normal Mode Analysis was performed on 73 active conformation structures in Ser/Thr/Tyr kinase superfamily. Categories of kinases with progressive evolutionary divergence, viz. (i) Same kinase with many crystal structures, (ii) Within-Subfamily, (iii) Within-Family, (iv) Within-Group, and (v) Across-Group, were analyzed. We identified a flexibility signature conserved in all kinases involving residues in and around the catalytic loop with consistent low-magnitude fluctuations. However, the overall structural fluctuation profiles are conserved better in closely related kinases (Within-Subfamily and Within-family) than in distant ones (Within-Group and Across-Group). A substantial 65.4% of variation in flexibility was not accounted by variation in sequences or structures. Interestingly, we identified substructural residue-wise fluctuation patterns characteristic of kinases of different categories. Specifically, we recognized statistically significant fluctuations unique to families of protein kinase A, cyclin-dependent kinases, and nonreceptor tyrosine kinases. These fluctuation signatures localized to sites known to participate in protein-protein interactions typical of these kinase families. We report for the first time that residues characterized by fluctuations unique to the group/family are involved in interactions specific to the group/family. As highlighted for Src family, local regions with differential fluctuations are proposed as attractive targets for drug design. Overall, our study underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Proteins 2016; 84:957-978. © 2016 Wiley Periodicals, Inc.