Transferred Nuclear Overhauser Effect Research Articles

Analysis by NMR Spectroscopy of the Structural Homology between the Linear and the Cyclic Peptide Recognized by Anti-human Leukocyte Antigen Class I Monoclonal Antibody TP25.99*

The anti-human leukocyte antigen (HLA) class I monoclonal antibody (mAb) TP25.99 has a unique specificity since it recognizes both a conformational and a linear determinant expressed on the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains, respectively. Previously, we reported the identification of a cyclic and a linear peptide that inhibits mAb TP25.99 binding to the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains (S. A. Desai, X. Wang, E. J. Noronha, Q. Zhou, V. Rebmann, H. Grosse-Wilde, F. J. Moy, R. Powers, and S. Ferrone, submitted for publication). The linear X(19) and cyclic LX-8 peptides contain sequence homologous to residues 239-242, 245, and 246 and to residues 194-198, respectively, of HLA class I heavy chain alpha(3) domain. Analysis by two-dimensional transfer nuclear Overhauser effect spectroscopy of the induced solution structures of the linear X(19) and cyclic LX-8 peptides in the presence of mAb TP25.99 showed that the two peptides adopt a similar structural motif despite the lack of sequence homology. The backbone fold is suggestive of a short helical segment followed by a tight turn, reminiscent of the determinant loop region (residues 194-198) on beta(2)-mu-associated HLA class I heavy chains. The structural similarity between the linear X(19) and cyclic LX-8 peptides and the lack of sequence homology suggests that mAb TP25.99 predominantly recognizes a structural motif instead of a consensus sequence.

Structure, modelling, and molecular dynamics studies of the inhibition of protein tyrosine phosphatase 1B by sulfotyrosine peptides

The protein tyrosine phosphatases comprise a class of enzymes that are crucial for the regulation of a number of cellular processes. Because of this, they are attracting increasing attention, not only as legitimate therapeutic targets, but also because of their relationship to many fundamental cellular processes. Certain sulfotyrosine peptides derived from casein are known to be good inhibitors of the protein tyrosine phosphatase, PTP1B. In this study, NMR transfer nuclear Overhauser effect studies have been used to ascertain the bound-state conformation adopted by the 12-amino acid residue casein-derived peptide, CAS200 (NANEEE(sY)SIGSA) and N-terminal truncated forms of this peptide, CAS203 and CAS205. Each of the peptides were found to bind in an extended beta-strand conformation. Extensive molecular modelling and molecular dynamics simulations of the PTP1B/peptide complexes, in a fully hydrated model, allowed a detailed description of the potential sources of the binding interactions to be developed. In agreement with the NMR studies, the modelling provided a picture of binding of CAS200 in which only the central (E203-I208) residues contributed significantly to the binding while the 3 N-terminal and 3 C-terminal residues were quite fluxional. Critical cationic surface residues, lying near to, but outside the active site pocket were the source of strong stabilizing forces that complemented the stabilizing interactions of the active site pocket. Electrostatic, hydrophobic, and hydrogen bonding interactions, in a residue specific manner, were all found to make significant contributions to the binding of these inhibitors.

Bound conformations for ligands for G-protein coupled receptors

The conformation of the C-terminus of the α-subunit of transducin, the G-protein of vision, has been determined by transfer NOE when bound to activated (MII) rhodopsin. One hundred three new NOE constraints are apparent when light is shown on a mixture of rhodopsin bilayers and the undecapeptide. Analogs of the α-peptide with covalent constraints were designed restricting the bound conformation; they stabilize MII thus supporting the deduced structure. The NMR structure of a complex of the intracellular loops of rhodopsin facilitates docking of the α-peptide and also shows proximity of residues known by mutational analysis to interact to generate the activated rhodopsin-transducin interface. This constrains the location of transmembrane helices in the structure of activated rhodopsin. Methods for the prediction of affinity have been used to estimate the relative binding constants of peptide analogs with the loop complex and show strong correlation with experimental data. Various models of the rhodopsin-transmembrane helical segments have been computationally fused with distance geometry to determine the overall model which best fits the experimental data on the rhodopsin-transducin interface.

Preparation of ( R)- and ( S)-1-adamantyl-9-anthrylmethanol. Conformational study and their behaviour as chiral solvating agents

Enantiopure forms of 1-adamantyl-9-anthrylmethanol were prepared and a conformational study was carried out by MM and NOE transfer methodologies. The absolute configuration was determined by combining dynamic NMR and theoretical calculations of the carbamate derivatives. The activity as a chiral solvating agent was tested in the presence of oxygenated functional groups.

Synthesis of inner core antigens related to Chlamydia, Pseudomonas and Acinetobacter LPS

Chemical syntheses of inner core determinants have been performed to provide defined artificial antigens (BSA-glycoconjugates) for characterization of monoclonal antibodies directed against important epitopes residing in the inner core of bacterial lipopolysaccharides. Efficient block synthesis of Kdo oligosaccharides has been employed to prepare the allyl glycoside [5] corresponding to the Chlamydia-specific Kdo trisaccharide epitope, to be used in crystallization and NMR (transfer NOe) experiments. Human pathogenic strains of Pseudomonas aeruginosa of RNA group I contain a highly phosphorylated heptose region with a 7- O-carbamoyl L- glycero-D- mannoheptose moiety which may be exploited as immunochemical marker for pathogenic Pseudomonas species. The 7- O-carbamoyl-substituted heptoside [12] as well as the disaccharides 7- O-carbamoyl-L- gro-α-D- manHep p- (1→3)-L- gro-α-D- manHep p-(1→O-Allyl) [23] and α-D-Gal pNAc-(1→3)-L- gro-α-D- manHep p-(1→O-Allyl) [30] were synthesized via regioselective formation of a 6′,7′- O-carbonate group followed by ring opening with NH3/NH4HCO3 to give the 7- O-carbamate in high yields. Finally, glycosides of the Kdo-isosteric D- glycero-D- talo-2-octulosonic acid (Ko) occurring in Acinetobacter spp. have been prepared via intermediate orthoester formation and TMSO-triflate-catalyzed rearrangement into α-ketosides. Coupling with a Kdo bromide donor and deblocking afforded the disaccharide α-Kdo-(2→4)-α-Ko-(2→O-Allyl) [43].

Characterization of the Human Cytomegalovirus Protease As an Induced-Fit Serine Protease and the Implications to the Design of Mechanism-Based Inhibitors

The conformational properties of the N-tert-butylacetyl-l-tert-butylglycyl-l-Nδ,Nδ-dimethylasparagyl-l-alanyl methyl ketone (MK) 1 and its terminal N-isopropylacetyl analogue 2 were investigated. Whereas these compounds are weak (mM IC50 range) inhibitors of the human cytomegalovirus (HCMV) protease, their activated carbonyl analogues are >1000-fold more potent (e.g., trifluoromethyl ketone 3, IC50 = 1.1 μM). A combination of NMR techniques demonstrated that MK 2 exists in solution as a relatively rigid and extended peptide structure and that the bulky side chains, notably the P3 tert-butyl group, greatly contribute to maintaining this solution conformation. Furthermore, transferred nuclear Overhauser effect (TRNOE) studies provided an enzyme-bound conformation of MK 2 that was found to be similar to its free solution structure and compares very well to the X-ray crystallographic structure of a related peptidyl inhibitor complexed to the enzyme. The fact that ligands such as MK 2 exist in solution in the bioactive conformation accounts, in part, for the observed inhibitory activity of activated ketone inhibitors bearing comparable peptidyl sequences. Comparison of the X-ray structures of HCMV protease apoenzyme and that of its complex with a related peptidyl α-ketoamide inhibitor allowed for a detailed analysis of the previously reported conformational change of the enzyme upon complexation of inhibitors such as 1 and 3. The above observations indicate that HCMV protease is a novel example of a serine protease that operates by an induced-fit mechanism for which complexation of peptidyl ligands results in structural changes which bring the enzyme to a catalytically active (or optimized) form. Kinetic and fluorescence studies are also consistent with an induced-fit mechanism in which a considerable proportion of the intrinsic ligand-binding energy is used to carry out the conformational reorganization of the protease. Issues related to the rational design of both mechanism- and nonmechanism-based inhibitors of HCMV protease, notably in light of the peptidyl ligand-induced optimization of its catalytic functioning, are discussed.

Bioaffinity NMR Spectroscopy: Identification of an E-Selectin Antagonist in a Substance Mixture by Transfer NOE

Unambiguous identification of the E-selectin antagonist 1 in a mixture of potential E-selectin ligands is possible with a bioactivity assay that is based on transfer NOE. This is remarkable, since the compounds present in the mixture have similar structures and their 1H NMR signals overlap extensively.

Bioaffinity NMR Spectroscopy: Identification of an E-Selectin Antagonist in a Substance Mixture by Transfer NOE

Bioaffinity NMR Spectroscopy: Identification of an E-Selectin Antagonist in a Substance Mixture by Transfer NOE

Conformational analysis of josamycin, a 16-membered macrolide free in solution and bound to bacterial ribosomes

The potent 14-membered macrolide antibiotics displayed a strong NMR response in the TRNOESY experiments whereas their metabolites which do not retain antimicrobial activity gave essentially blank TRNOESY spectra. These TRNOESY experiments are here extended to a macrolide (josamycin) belonging to the 16-atom macrolide class in order to compare responses according to the macrocycle size. Analysis of transferred nuclear Overhauser effect (TRNOE) experiments resulted in a set of constraints for all proton pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain a bound structure compatible with the experimental NMR data. This study allowed us to identify the binding structure at the ribosome for the active 16-membered macrolide. The different conformations existing in solution for this antibiotic in the free state will be compared and correlated with the binding conformation at the ribosome obtained with TRNOE experiments so as to establish a structure–activity relationship. The comparative results indicate that one conformation (called S3) pre-existing in the conformational equilibrium of macrocycle in solution and close to the major one S5, is preferred in the bound state.

Binding of nucleotides by the mitochondrial ADP/ATP carrier as studied by 1H nuclear magnetic resonance spectroscopy.

Nucleotide binding to the cytosolic binding site of the mitochondrial ADP/ATP carrier (AAC) was studied by 1H-nuclear magnetic resonance spectroscopy. Binding (as opposed to translocation) could be identified as a result of the rapid ligand on/off kinetics, using the cytosolic side specific inhibitor carboxyatractyloside (CAT) for the distinction from nonspecific interactions. The off rate constant of the nonhydrolyzable ATP analogue AMP-PCP was more than 3 orders of magnitude larger than the transport rate. The nucleotides adopt an anti conformation in the carrier binding site as shown by measurements of the transferred nuclear overhauser effect (TRNOE). A thermal transition around 14 degreesC that had been previously detected in transport studies [Klingenberg, M., Grebe, K., and Appel, M. (1982) Eur. J. Biochem. 126, 263-269] was reflected by the inhibitor sensitive line broadening, indicating that this transition also affects nucleotide binding. Nucleotide monophosphates were employed to study the relation between nucleotide structure and affinity, using selective excitation, sample spinning with digital suppression of spinning sidebands, and line shape simulation. The binding of purines depends on the distribution of the electrical potential and on the position of ring substituents, while pyrimidines are barely recognized at all by the AAC. It is also shown that the photocleavable "caged" derivatives are more tightly bound than the original nucleotides. A two step model of carrier catalysis will be discussed on the basis of these results.

15N-edited Three-Dimensional NOESY-HMQC with Water Flipback: Enhancement of Weak Labile1H Resonances of Protein Side Chains Contacting DNA

Two pulse sequences are described that employ a modified water flipback technique to enhance the signal intensity of weak side chain resonances at the protein–DNA interface of the vnd/NK-2 homeodomain/DNA complex in an15N-edited three-dimensional NOESY-HMQC spectrum. The pulse sequences presented employ water flipback pulses at the beginning of the NOESY mixing time, optimizing the direct NOE transfer of magnetization from the water to the protein by maximizing thez-component of the water magnetization. In one of the pulse sequences, radiation damping during the the indirect1H and15N evolution times is suppressed. A modified version of the WATERGATE water suppression technique is employed during the HMQC portion of the experiment. The signal enhancement is demonstrated for the resonances of the side chain amide of Asn51, an invariant homeodomain residue whose contact with the DNA is critical for binding. An ancillary advantage of the experiment is the ability to observe NOE transfer of magnetization from water. The information present in the water resonance plane of the three-dimensional spectrum is illustrated in a comparison with the corresponding HMQC spectrum of the protein/DNA complex.

Determination of Binding Conformations of Drugs to Human Serum Albumin by Transferred Nuclear Overhauser Effect Measurements and Conformational Analyses Using High-Temperature Molecular Dynamics Calculations

□ The binding conformations of oxyphenbutazone (OXY), Ne-dansyl-L-lysine (DNS-LYS), and furosemide (FU) to human serum albumin (HSA) have been investigated by molecular dynamics (MD) calculations and transferred nuclear Overhauser effect (TRNOE) measurements. We have combined distance information obtained from the Conformational Analyzer with Molecular Dynamics And Sampling (CAMDAS) calculation and experimental NOE spectroscopy measurements to determine a “binding conformation” for each drug which binds to site I of HSA. For OXY, only one conformer (conf9) among the conformer set generated by MD calculation satisfied the distance restraint conditions obtained from TRNOE measurements. For DNS-LYS and FU, 17 and 5 conformers satisfied distance restraint conditions, respectively. The structure of conf9 of OXY was taken as a “template” to choose binding conformers for DNS-LYS and FU. By fitting the “template” to the 17 conformers of DNS-LYS and 5 conformers of FU, we could efficiently obtain one binding conformer for DNS-LYS (conf144) and FU (conf26). It is suggested from the feature of the binding conformation that the three-dimensional location of a hydrophobic aromatic ring, alkyl chain, and electronegative functional group is important for binding to site I of HSA. This method, which combines MD calculations and NOE information, is thought to be effective for determining the binding conformation of drugs to HSA.

Transferred nuclear Overhauser effect study of macrolide–ribosome interactions: correlation between antibiotic activities and bound conformations

The study of macrolide–ribosome interactions has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). A new medically important macrolide antibiotic, roxithromycin, with the replacement of the 9-keto group in erythromycin by a 9-oxime chain, was studied in the complex state with the bacterial ribosome. Analysis of transferred nuclear Overhauser effect (TRNOE) experiment resulted in a set of constraints for all protons pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrate in solution indicate that only one specific conformation is preferred in the bound state while in the free state the sugar ring moities were relatively disordered. The bioactive macrolide antibiotics studied roxithromycin and erythromycin which displayed a strong NMR response, are metabolized in RU39001 and erythralosamine respectively which do not retain antimicrobial activity. The inactive major metabolites were used to define if TRNOEs observation may be characteristic of a biological activity. These control experiments gave essentially blank TRNOESY spectra. This study shows that Mg 2+ does not play a direct role for the low affinity binding site studied by TRNOE what is in agreement with an hypothesis of two distinct binding levels, with a low affinity binding level necessary for the tight binding one.

Comparison of the Bioactive Conformations of Sialyl LewisX and a Potent Sialyl LewisX Mimic

For the rational design of drugs that inhibit the interaction between E-selectin and sialyl LewisX, structural information on bound inhibitors is required. The bioactive conformation of the potent sialyl LewisX mimic 1 was therefore determined by transfer NOE NMR spectroscopic methods.

Transferred nuclear Overhauser effect spectroscopy study of a peptide from the PapG pilus subunit bound by the Escherichia coli PapD chaperone

Interaction of the Escherichia coli PapD chaperone with the synthetic peptide PapG308-314 (Thr–Met–Val–Leu–Ser–Phe–Pro), corresponding to the seven C-terminal residues of the PapG pilus subunit, was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The observation of cross-peaks corresponding to either intraresidue or sequential C αH/NH and C βH/NH TRNOEs and the absence of sequential NH i/NH i+1 TRNOEs indicate that the peptide binds to PapD in an extended conformation. In addition, line-broadening effects gave information of the peptide's mode of interaction with PapD. These observations were in excellent agreement with a recent crystal structure of a PapG peptide complexed with PapD.

Screening mixtures for biological activity by NMR.

Development of the new drugs often involves the screening of compound libraries for biological activity. Currently, the biologically active component can only be identified if either a pure compound is being tested or if the components of a mixture are spatially separated, for example, on beads. Here, we present an NMR technique based on the transferred nuclear Overhauser effect (transfer NOE) that allows identification and structural characterization of biologically active molecules from a mixture. As an example we demonstrate that from mixtures of oligosaccharides only alpha-L-Fuc-(1-->6)-beta-D-GlcNAc-OMe binds to Aleuria aurantia agglutinin. The sign of transferred NOEs is opposite to NOEs of small molecules that do not bind to the protein and, thus, an unequivocal identification of molecules with binding activity is possible. Normally, the selection of bound ligands is further facilitated in that the absolute intensity of transfer NOEs is much greater than that of NOEs of non-binding molecules. In addition, transfer NOEs provide information on the three-dimensional structure of the ligands in the bound state. Therefore, measuring transfer NOEs of mixtures of small molecules in the presence of large molecules, like proteins, should significantly enhance the options for screening mixtures of compounds for biological activity.

On the Interaction Between p53 and MDM2: Transfer NOE Study of a p53-Derived Peptide Ligated to MDM2

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTOn the Interaction Between p53 and MDM2: Transfer NOE Study of a p53-Derived Peptide Ligated to MDM2M. J. J. Blommers, G. Fendrich, C. García-Echeverría, and P. ChêneView Author Information Physics, Core Drug Discovery Technologies, and Oncology Research, Ciba-Geigy AG, P.O. Box, CH-4002 Basel, Switzerland Cite this: J. Am. Chem. Soc. 1997, 119, 14, 3425–3426Publication Date (Web):April 9, 1997Publication History Received23 September 1996Published online9 April 1997Published inissue 1 April 1997https://doi.org/10.1021/ja9633171Copyright © 1997 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views200Altmetric-Citations17LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (83 KB) Get e-AlertsSUBJECTS:Cancer,Chemical calculations,Chemical structure,Monomers,Peptides and proteins Get e-Alerts

Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewis(x) bound to E-selectin.

The conformation of the sialyl Lewis(x) tetrasaccharide bound to E-selectin was previously determined from transfer NOE (trNOE) experiments in conjunction with a distance-geometry analysis. However, the orientation of the tetrasaccharide ligand in the binding site of E-selectin is still unknown. It can be predicted that the accurate quantitative analysis of all trNOEs, including those originating from spin diffusion, is one key to analyze the orientation of sialyl Lewis(x) in the binding pocket of E-selectin. Therefore, we applied homonuclear 3D NMR experiments and 1D analogs to obtain trNOEs that could not unambiguously be assigned from previous 2D trNOESY spectra, due to severe resonance-signal overlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a 1D trNOESY-TOCSY experiment of the sialyl Lewis(x)/E-selectin complex furnished new interglycosidic trNOEs and provided additional information for the interpretation of trNOEs that have been described before. A 2D trROESY spectrum of the sialyl Lewis(x)/E-selectin complex allowed one to identify the amount of spin-diffusion contributions to trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysis of spin-diffusion pathways, was obtained, creating a basis for a quantitative analysis of trNOEs in the sialyl Lewis(x)/E-selectin complex.

Molecular modeling of the neurophysin I/oxytocin complex.

Neurophysins I and II (NPI and NPII) act in the neurosecretory granules as carrier proteins for the neurophyseal hormones oxytocin (OT) and vasopressin (VP), respectively. The NPI/OT functional unit, believed to be an (NPI/OT)2 heterotetramer, was modeled using low-resolution structure information, viz. the C alpha carbon atom coordinates of the homologous NPII/dipeptide complex (file 1BN2 in the Brookhaven Protein Databank) as a template. Its all-atom representation was obtained using standard modeling tools available within the INSIGHT/Biopolymer modules supplied by Biosym Technologies Inc. A conformation of the NPI-bound OT, similar to that recently proposed in a transfer NOE experiment, was docked into the ligand-binding site by a superposition of its Cys1-Tyr2 fragment onto the equivalent portion of the dipeptide in the template. The starting complex for the initial refinements was prepared by two alternative strategies, termed Model I and Model II, each ending with a approximately 100 ps molecular dynamics (MD) simulation in water using the AMBER 4.1 force field. The free homodimer NPI2 was obtained by removal of the two OT subunits from their sites, followed by a similar structure refinement. The use of Model I, consisting of a constrained simulated annealing, resulted in a structure remarkably similar to both the NPII/dipeptide complex and a recently published solid-state structure of the NPII/OT complex. Thus, Model I is recommended as the method of choice for the preparation of the starting all-atom data for MD. The MD simulations indicate that, both in the homodimer and in the heterotetramer, the 3(10)-helices demonstrate an increased mobility relative to the remaining body of the protein. Also, the C-terminal domains in the NPI2 homodimer are more mobile than the N-terminal ones. Finally, a distinct intermonomer interaction is identified, concentrated around its most prominent, although not unique, contribution provided by an H-bond from Ser25 O gamma in one NPI unit to Glu81 O epsilon in the other unit. This interaction is present in the heterotetramer (NPI/OT)2 and absent or weak in the NPI2 homodimer. We speculate that this interaction, along with the increased mobility of the 3(10)-helices and the carboxy domains, may contribute to the allosteric communication between ligand binding and NPI dimerization.

Molecular Modelling of the Neurophysin II/Vasopressin Complex

AbstractNeurophysins I and II (NPI and NPII) serve in the neurosecretory granules as carrier proteins to the neurophyseal hormones oxytocin (OT) and vasopressin (VP), respectively. The (NPII/VP)2 heterotetramer, believed to be the smallest functional unit, was modelled using a low‐resolution structure information, viz. the Cα‐carbon atom coordinates of the homologous NPII/dipeptide complex (file 1BN2 in the Brookhaven Protein Data Bank) and a recently proposed (from the transfer NOE experiment) NPI‐bound OT structure as template. An all‐atom representation was obtained using standard modelling tools available within the SYBYL suite of programs (by Tripos, Inc.). The starting complex was elaborated for further refinements by two alternative strategies, termed Model I and Model II. Model I consisted of a constrained simulated annealing (CSA) protocol while Model II consisted of carefully elaborated sets of constrained minimizations. Both Models were terminated with a ∼ 100 ps molecular dynamics (MD) in water using the AMBER 4.1 force field. The free homodimer NPII2 was obtained by the removal of the two VP subunits from their sites and elaborated according to Model I only. Both models have lead to structures similar to the solid state NPII/dipeptide complex and the recently published the NPII/OT (cross)complex. Nonetheless, Model I as one implementing constrained simulated annealing and thus less amenable to personal bias, is further recommended as a method of choice for the preparation of the starting all‐atom structures for MD. The MD simulations indicated that both in the homodimer and in the heterotetramer the 310 helices display increased mobility relative to the remainder of the protein. Also, the C‐terminal domains in the (NPII/VP)2 heterotetramer are more mobile than the N‐ terminal ones. In addition to a pair of distinct inter‐monomer attractive contributions, provided by H‐bonds from Ser25 O7 of one NP unit to Glu81 Oϵ of the other, nonbonded attractive interactions involving binding site of unit 1(2) and the inter‐mononer interface of unit 2(1) are clearly seen. We speculate that both the increased mobility of the 310 helices and carboxyl domains, as well as these interactions may contribute to the allosteric communication between the ligand binding and the NPII dimerization.