Distant site mutations in clinical TEM β-lactamase variants enhance non-covalent binding to ceftazidime: Insights from biophysical and in silico investigations

Distant site mutations in clinical TEM β-lactamase variants enhance non-covalent binding to ceftazidime: Insights from biophysical and in silico investigations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Distant site mutations in clinical TEM β-lactamase variants enhance non-covalent binding to ceftazidime: Insights from biophysical and in silico investigations Sandip Kumar Mukherjee, Padmaja Prasad Mishra, Mandira Mukherjee This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7504842/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose This study investigated the bioactive interactions of ceftazidime (CAZ) to TEM β-lactamase variants with distant site mutations isolated from clinical settings to explore the cause of their selection and dissemination due to empirical use of β-lactams. Methods Binding interactions of CAZ with wild-type and mutant (M184, M203, M210) TEM β-lactamases were recorded by UV–Vis absorption and fluorescence spectra (280–600 nm) at 298–311 K with inner filter correction. Binding constants were determined using Stern–Volmer and modified Stern–Volmer equations. Fluorescence lifetime, quantum yield and anisotropy measurements ascertained energy transfer and microenvironmental changes. Structural alterations and interaction energetics were evaluated using circular dichroism, Raman spectroscopy, Förster resonance energy transfer and Molecular dynamics simulation (100 ns) study. Results Biophysical experimentation indicated facilitated binding of CAZ to the active site of the mutants than the wild type. The CAZ-TEM β-lactamase mutant interactions were predominantly hydrophobic compared to H-bonding and van der Waals forces in the CAZ-wild type complex. Additionally, structural alteration to justify more rigid binding of CAZ to the mutants in contrast to the wild type enzyme was established in silico . Acquisition of distant site mutations with respect to the active site of TEM β-lactamases rendered conformational flexibility to accommodate CAZ was evidenced. Conclusion Therefore, this study established that selection of far site mutations in TEM β-lactamases can indirectly contribute to β-lactam resistance by optimizing substrate binding dynamics in response to rampant usage of the antibiotics and provided a new dimension towards future drug development. TEM β-lactamase mutants absorbance spectroscopy fluorescence quenching fluorescence anisotropy Van't Hoff plot Molecular dynamics simulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Persistence of bacterial resistance against antimicrobial agents is a global health threat [ 1 ]. Production of active site serine β-lactamase enzymes is the major mechanism of antibiotic resistant in uropathogenic Escherichia coli (UPEC) that catalyze the hydrolysis of β-lactam antibiotics. [ 2 – 4 ]. Currently, based on Ambler classification scheme, β-lactamases can be grouped into four classes A, B, C and D. Class A, C, and D are serine β-lactamases (non-metallo) that utilize the serine residue to catalyze the hydrolysis of antibiotics, whereas class B enzymes are metallo-β-lactamases and need zinc ions in the active site to attack the β-lactam ring [ 4 ]. .In the case of Class A β-lactamases which is prime concerned for the clinicians due to its wide range of hydrolyzing activity against antibiotics mostly against β-lactam antibiotics where activation of catalytic Ser70 conserved residue initiates nucleophilic attack on the β-lactam carbonyl carbon and opening of the β-lactam ring to form a covalently linked acyl-enzyme intermediate and ultimately the attack leads to deacylation of the acyl-enzyme species resulting inactivation of antibiotic and regeneration of the free enzyme [ 4 ].TEM1 β-lactamase was identified as one of the most evolved Class A β-lactamases in hospital infection, mediated by plasmid found in E. coli resistant to antibiotics and also considered as the precursor of the TEM enzyme family with three major types of activity; the broad-spectrum β-lactamases, the extended spectrum β-lactamases (ESBLs) and the inhibitor resistant β-lactamases. The emergence of resistance mediated by this TEM enzyme family were most explored β-lactamases till date and it was found that their extensive structural adjustment by their sustainable mutational modifications led to the generation of distinct multiple variants with in-depth structural diversity [ 5 ]. To overcome β-lactam resistance extensive studies were carried out against the acylation mechanism and two types of β-lactams were introduced such as extended spectrum cephalosporins; and β-lactamase inhibitors, such as clavulanic acid, sulbactam and tazobactam [ 6 ]. Furthermore, the structural alterations in Class A β-lactamases impair the antibacterial activities of the inhibitors by covalent interaction between the active site residues in the former with the inhibitory molecules resulting in resistance against them [ 7 ]. Till date ~ 200 TEM-variants have been reported that confers resistance to novel β-lactams and their inhibitor combinations which were introduced in the past three decades [ 8 , 9 ]. Studies from the different part of the world showed that the indiscriminate use of β-lactam-β-lactamase inhibitor combination has increased the number of TEM β-lactamase variants; the extended-spectrum (ES) and inhibitor-resistant phenotypes (IR) [ 10 , 11 ]. Previous reports showed that mutation or substitution in the active site Ω-loop region of TEM β-lactamase not only increased the resistance phenotype against cephalosporins but also increased the structural flexibility imparting enlargement of the active site pocket loop that can accommodate newer β-lactams irrespective of their -R group thus decreasing the activity of oxyimino-cephalosporin, ceftazidime (CAZ), and all other β-lactam-β-lactamase inhibitors [ 3 , 4 ]. Fluorescence techniques were widely used as one of the important biophysical application that paved sensitivity, reliability and rapidity in the field of drug-protein interactions study [ 12 , 13 ]. There are not many Indian studies that described existence and characterization of TEM β-lactamase mutants that harbor mutations at sites other than the active site of the enzyme isolated from clinical origin as a result of random drug administration. An earlier study from our laboratory, demonstrated identification and characterization of three novel TEM β-lactamase mutants; pUE184TEM (M184), pUE203TEM (M203), pUE210TEM (M210) from clinical uropathogenic E. coli isolates with mutation at residues other than those that constituted the active site of the enzymes [ 14 ]. In the present study bioactive interactions of CAZ with the mutant β-lactamases under physiological conditions was investigated. The role of distant site mutations on the conformational flexibility in the mutants to accommodate CAZ was explored by using a collection of biophysical and in silico methods to obtain an insight into selection and dissemination of the resistant β-lactamases in response to uncontrolled β-lactam administration. Materials and Methods Absorbance Spectroscopy Absorbance spectral measurement was recorded in UV–vis Spectrometer at a scan rate of 480 nm/min with 1 cm-path-length quartz cuvette in the visible wavelength range of 200-800nmat different temperatures (298K, 303K, 308K, 311K).The titrations of the experiment were conducted on 5 µM wild type TEM βlactamase and its variants (M184, M203, M210) [14)] in presence of ascending concentration of CAZ (5 µM-20µM) at respective temperatures. The inner filter effect was eliminated using the relationship; I cor = I obs e (Aex+Aem)/2 ,where the corrected fluorescence intensity is denoted as I corr , and observed fluorescence intensity is denoted as I obs . The A ex and A em denote absorbance values at excitation and emission wavelengths, respectively [15]. Fluorescence Spectroscopy Fluorescence emission spectrum was recorded in the range of 280–600 nm using Hitachi F-7000 Spectrofluorimeter with excitation at 295 nm. Both the excitation and emission bandwidth were fixed at 5 nm. Quenching experiment was carried out in presence of increasing concentration of CAZ (5 µM-20µM) solution at different temperatures (298K, 303K, 308K, 311K) to 5 µM concentration of the wild type and the mutant βlactamases. Static fluorescence quenching was determined by using the Stern–Volmer (SV) equation from steady state measurements F 0 /F = 1 + K sv (Q), where F 0 and F denote the steady-state fluorescence emission intensities in the absence and in presence of CAZ. K SV is the Stern–Volmer constant and (Q) is the concentration of the quencher. Collisional or dynamic fluorescence quenching was determined using the modified SternVolmer equation; log ((F 0 -F)/F)) = logK a + n log (Q), where K a denoted the binding constant and n the number of binding sites. Fluorescence lifetime measurements The fluorescence lifetime(τ) measurements determine energy transfer at the molecular level between interacting species. Time correlated single photon counting (TCSPC) was used to measure the fluorescence life times of wild type and mutant βlactamases in gradually increasing concentration of CAZ (0 µM-20µM) in sodium phosphate buffer (pH 7.4) at 298K [16]. Quantum yield and fluorescence anisotropy The quantum yield (Φ) or quantum efficiency of CAZ at 5µM concentration in sodium phosphate buffer (pH 7.4) was recorded by monitoring the fluorescence emission intensity in presence of increasing concentrations of the wild type and mutant β lactamases (0–20 µM)at 298K [ 17 ]. The fluorescence anisotropy (r) measurement of CAZ at 5µM concentration in 5mM sodium phosphate buffer (pH 7.4) was recorded against addition of ascending concentration of wild type and mutant proteins (5µM-20µM) in Fluromax Spectrofluorometer. All measurements were conducted at 298K at 3 minutes of interval. Thermodynamic properties Van't Hoff isotherm equation was used to determine the thermodynamic parameter (kinetic and equilibrium properties) that govern the interaction between the ligands (CAZ) and the proteins (TEM and its mutants). The values of ∆H 0 and ∆S 0 were obtained from the lnK vs. 1/T plot, where ∆G 0 , ∆H 0 , and ∆S 0 are depicted as standard enthalpy change, standard free energy change, and standard entropy change respectively. Circular Dichroism (CD) Spectroscopy Far and Near UV-CD spectra was recorded at room temperature for βlactamases (TEM and its mutants) in presence of ascending concentration of ceftazidime ranges from 0 µM-20µM solutions using JASCO-J815 spectrometer with a temperature controller at 298 K in a quartz cuvette of 1 mm path length. Each final spectrum was recorded as an average over three scans. The far and near-UV-CD region was recorded as 250–360 nm and 360 nm to 500 nm.CD results were expressed as mean residue elasticity (MRE) in degree cm 2 mol − 1 . The secondary structures (α-helix, β-sheet, random coils) were calculated from respective MRE values [18]. Raman spectroscopy Raman scattering was recorded for βlactamases (TEM and its mutant variants) in presence of ascending concentration of ceftazidime ranges from 0µM-20µM. The spectral range was taken for500–4000 cm − 1 with an integration time of 20 s using a 670 nm red laser excitation, at 10 mW power [19]. Förster resonance energy transfer (FRET) computation FRET involves a distance dependent physical method of non-radiative transfer of energy between an excited fluorophore (donor) and another fluorophore (acceptor) by intermolecular dipole-dipole interactions. The energy transfer efficiency (E) signified the fraction of photons absorbed by the donor moiety which was transferred to the acceptor. E = 1-(F/F 0 ) = R 0 6 /(R 0 6 + r 6 ), where, r represented the distance between the donor and acceptor and R 0 represented the critical energy transfer distance, at which 50% of the excitation energy was transmitted from the former to the latter. R 0 was evaluated from the following equation; R 0 6 = 8.79 x 10 − 25 K 2 N −4 ϕ, where K 2 was the spatial orientation factor of the altered dipoles of the acceptor and the donor, N represented the refractive index of the medium, ϕ denoted the fluorescence quantum yield of the donor in absence of the acceptor [ 20 ]. Molecular dynamics simulation Molecular dynamics and simulation were performed on the TEM β-lactamase (1ZG4) and its variants [ 14 ] in presence of CAZ to determine the binding stability of the protein–drug docked complex using Desmond v3.6 Package. Topology of the protein molecules were generated by optimized potentials for liquid simulation (OPLS) force field. Simple point charge (SPC) water model was used to perform the protein–drug complexes Simulation with a distance of 10 Å between the complex and the periodic box. System was neutralized by adding Na+/Cl − ions and placed randomly in the solvated system. To minimize and relax the protein/protein − drug complex under NPT ensemble, the default protocol of Desmond was used followed by total of nine stages among which two minimization and four short simulation (equilibration phase) steps was involved before starting the actual production time. System temperature and pressure was 300 K and 1 atmosphere, respectively, using Nose − Hoover temperature coupling and isotropic scaling, and the simulation was performed for 100 ns NPT production simulation and saving the configurations at 10 ns intervals [ 21 ]. Simulation trajectory analysis Simulation trajectory files were analyzed using Desmond module programs i.e. simulation quality analysis (SQA), simulation event analysis (SEA), and simulation interaction diagram (SID) to calculate energies, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (Rg) and the secondary structure elements (SSEs) which pave the protein structure stability. SQA was used to qualitatively validate the system stability throughout the simulated length of chemical time for the given temperature, pressure, and volume of the total simulation box, SEA was used to analyze each frame of the simulated trajectory output, whereas total SSE change in the protein structure in presence of the drug during the simulation time was estimated using SID [ 22 , 23 ]. Results Spectroscopic measurements UVVis absorption spectra of wild type and mutant βlactamases (Fig. 1A-D) were recorded in presence of increasing concentrations of ceftazidime, at 298K. All protein showed a broad band at 280 nm while ceftazidime had a strong absorbance at 267 nm and a moderate absorbance at 310 nm. With gradual increase of CAZ concentration, the absorbance spectra of wild βlactamase showed enhancement of absorption intensity. The possibility of ground state complex formation couldnot be ruled out from the above absorption spectra. Additionally, from Fig. S1 it was observed that the spectra of the mixture of CAZ and wild type βlactamase ( curve 5 ) was much intense than intensity of spectra corresponding to the summation of the two absorption bands of the individual reacting components ( curve 3 ). The curve 4 obtained by subtracting the curve 3 from the curve 5 indicated formation of ground state complex between the two species. The UV-Vis absorption spectra of the wild type and mutant β lactamases in presence of CAZ were recorded at 303 K, 308 K and 311 K respectively which indicated that the proteins-CAZ complexes were thermally stable ( Fig. S2 ). The fluorescence spectra of wild type and mutant βlactamases at pH 7.5in the presence of 5, 10, 15, 20µM CAZ at 298K showed strong fluorescence intensity at 340 nm upon excitation at 280 nm which gradually decreased upon addition of CAZ with a slight red shift at 298 K (Fig. 1E-H, Fig. S3 ). Similar comparable fluorescence spectra of the βlactamases at the different temperatures (303K.308K, 311K) were also recorded (data not shown). The quenching phenomenon exhibited in the fluorescence emission spectra of the proteins and protein –CAZ complexes were analyzed by Stern–Volmer (SV) plot. The non-linearity observed in the SV plot at 298K, 303K, 308K, 311K (Fig. 2A-D) indicated a combination of static (ground state) and dynamic (excited state) quenching which was validated by modified SV plots (Fig. 2E-H). Additionally, SV plots of quenching of the wild type in presence of increased concentration of CAZ at different temperatures indicated a gradual shift towards dynamic mode although mutants exhibited a decrease in their dynamic mode of quenching. Furthermore, fluorescence lifetime changes of the wild type and the mutant proteins in presence of different concentrations of CAZ also exhibited a steady decrease (Fig. 3A, Table 1). Fluorescence quantum yield and fluorescence anisotropy of CAZ Absolute fluorescence quantum yield was also analyzed for small molecule i.e. CAZ to measure changes in radiative rates from changes in nonradiative rates occurring in terms of interaction. The quantum yields of the CAZ molecule increased with the increase in concentration of proteins (wild and all three mutant βlactamases). The quantum yields of CAZ increased gradually from 0.08 to 0.27, 0.36, 0.36 and 0.37 for the wild type, M184, M203 and M210 βlactamase respectively (Table 2).Fluorescence anisotropy was also performed to measure the microenvironment around CAZ in terms of rotational diffusion and interactions. The anisotropy of the CAZ molecule was very low (r = 0.07). With increased concentration of the proteins (wild type and all three mutant βlactamases), fluorescence anisotropy of CAZ increased gradually. At 20µM concentration of CAZ a steady increase in r value from 0.18, 0.20, 0.21 to 0.24 in presence of the respective βlactamases (wild type, M184, M203, M210) respectively (Fig. 3B) Binding constants, number of binding sites and mode of binding The steady state fluorescence data were further evaluated by the binding affinity (K b ) of CAZ molecule to the equivalent sites of wild type and mutant βlactamases independently. For mutant βlactamases a progressive decrease of binding affinity (K b ) was observed with the increase of temperature whereas in case of wild type a vice versa affinity was observed in pH 7.5.(Fig. 4). The equilibrium interaction (such as, hydrogen bonds, van der Waals forces, electrostatic forces, and/or hydrophobic associations), between ligand (CAZ) and protein molecule were determined from the value of the thermodynamic parameter (∆G 0 , ∆H 0 , ∆S 0 )using van't Hoff isotherm equation from the lnK vs. 1/T plot. Significant negative ∆G 0 value, ∆H 0 0 indicated spontaneous interaction between the reacting components. Increase in negative ∆G 0 value with increase in positive ∆H 0 and ∆S 0 values in the mutantβlactamases indicated discrete predominance of hydrophobic interaction in contrary to the wild type βlactamase that exhibited van der Waals and H-bond interaction with high negative ∆H 0 and ∆S 0 values compared to less negative ∆G 0 value (Fig. 4, Table 3). Circular Dichroism (CD) Spectroscopy Conformational changes of wild and mutant βlactamases were further measured by CD spectroscopy in the absence and presence of CAZ at pH 7.5. Results showed that the mean residue ellipticity (MRE) of pure α-helix percentage of wild and mutant βlactamases were decreased upon binding of CAZ demonstrated secondary structure alternations of the proteins in far-UV-CD region (185–250 nm) with two characteristic dips at 209 nm and 222 nm for all the proteins respectively. Moreover, in the near-UV-CD region spectra (240–320 nm) of the wild type and mutant proteins showed comparable minor alterations of their tertiary structure ( Fig. S4 , Table 4) Raman spectrum of βlactamase proteins Raman intensity of wild type and mutant βlactamases (5µM) were measured in presence of ascending concentration of CAZ (5 µM-15 µM). Results showed a gradual increase of Raman intensity of βlactamases with the increase of the concentration of CAZ in comparison with the wild type protein. There are three regions (880 cm − 1 , the region of Tyr doublet due to H-Bonding),(1360 cm − 1 , CH2- CH2 deformation) and (1640–1680 cm − 1 , characteristics of amide peak) in the spectrum which indicated the secondary structure alternation due to molecular interaction. A small blue shift was also observed for the mutant β lactamases in comparison with the wild type due to secondary structure adjustment during interaction with CAZ. ( Fig. S5 ) Energy transfer between CAZ and β lactamases The absorbance spectrum of CAZ (excited at 267 nm) and fluorescence spectrum of the TEM 1 β- lactamase at 340 nm overlapped with each other which satisfied the requisite condition of FRET between the CAZ and the proteins (Fig. S6) . Moreover, from the Förster theory of energy transfer, the molecular interactions between the drug-protein complexes were evidenced from discrete values of the energy transfer efficiency (E); 0.47, 0.94, 0.79 and 0.82 for CAZ-wild type, CAZ-M184, CAZ-M203 and CAZ-M210 respectively which facilitated binding of CAZ to the mutants than the wild type. In silico study Earlier in silico study from our laboratory indicated effective binding of CAZ to the wild type and mutant β lactamases (Mukherjee S K et al, 2018). The stability and conformational changes of the β lactamase in presence of CAZ were analyzed by MD simulation study. The movement of atoms and molecules for 100 ns of simulation were monitored. In presence of CAZ, the wild type β lactamases (1ZG4) and M184 exhibited C-α backbone deviations at about ∼1.5 to 1.75 Å and ∼1.75 to 2.5 Å respectively throughout the simulation trajectory and an equilibrium was maintained after 40 ns for both. However, for M203 and M210 C-α backbone deviations from ∼1.5 to 2.5 and 1.75 to 2.5 Å with an elevation at 60 ns for the latter which reached equilibrium from 70 ns was observed. The C-α all residue deviations for M184 and M210 were ∼2.5 to 3.25 Å and ∼2.5 to 3.40 Å respectively with an elevation at 60 ns and equilibrium from 70ns in case of the latter. In contrast, M203 showed reduced Cα backbone deviation at 30 ns akin to wild type throughout the 100 ns simulation trajectory. However, M203 showed C-α all residue deviations from ∼2.25 to 3.0 in comparison to wild type in presence of CAZ (Fig. 5A-F) The residue level fluctuations (RMSF) of Cα backbone for M184 and M210 in presence of CAZ were moderately high (∼3.25 Å) in comparison to wild type at residues located between 50–80, 120–170, 200, and 225–250 respectively. However, a comparable pattern of fluctuation was observed for M203 and wild type except at residue 150. Similarly, RMSF for Cα side chain of the mutant proteins (M184, M203, M210) in presence of CAZ compared to wild type in presence of the drug showed similar pattern as the RMSF for Cα backbone (Fig. 5G-L). The compactness of protein-drug interaction was ascertained by Cα Radius of gyration (Rg) plot. The residue level Rg value fluctuation for wild type protein was within the range of ∼17.8 to 18.3 Å, whereas for mutant protein (M184) was within the range of ∼17.8 to 18.2 Å and reached equilibrium from 40 ns onward in presence of CAZ. Furthermore, in presence of the drug the Rg value fluctuations for M203 was ∼17.8 to 18.1 across the 100 ns simulation trajectory but for M210, the Rg value fluctuations was ∼17.9 to 18.6 Å with a decrease in fluctuation within the range of ∼18.2 to 18.4 Å from 60 ns onward to attain the equilibrium. The overall Rg analysis for compactness in the mutants in presence of CAZ showed lowest radius of gyration that demonstrated tight binding of CAZ to the mutant proteins in comparison to wild type (Fig. 5M-O). The protein-drug interactions were further monitored by estimation of secondary structure elements (SSE) composition (helices, strands, turns, loops) in the interacting proteins across the simulation trajectory throughout the 100 ns time frame. SSE for each amino acid residue of the mutants exhibited significant structural adjustment in comparison with wild type in presence of CAZ. In M184 increase of 310-helix in residue positions 100, 160, 220, turns in residue position 50, 90, 160, coils in position 140, 200 and π-helix in positions 210 to 225 was evidenced. In M203, the segment of the residues in positions 60–90 showed increase of 310-helix, coil structure at residues 150–170 and 225–240. M210 showed an increase of α-helix and coil with the absence of π-helix at positions 60–90 and 160–170 in comparison with the segment of residues in the wild-type with increase of 310-helix and increase of π-helix at residue position 210–225 respectively (Fig. 5P-T). Discussion Emergence of varied β-lactamases was reported which was the primary cause of β-lactam resistance in uropathogenic E. coli , the primary etiologic agent of urinary tract infection. These β-lactamases were found to harbor mutations which paved the possibility of effective binding of the different β-lactams and their inhibitor combinations to develop resistance. Therefore, studies on the protein-drug interactions have become increasingly important from a pharmaceutical perspective. Absorption spectroscopy study provide information on the binding modes of ligands with proteins pave the possibility of ground state complex formation between protein-ligand interfaces [ 15 ]. The individual UVVis absorption spectrum of the CAZ and TEM-1 βlactamase were markedly different to that of CAZ-βlactamase complex, indicating the formation of new complexes between CAZ and βlactamases. Appearance of isosbestic points at 339.4 nm, 360.2 nm, 341.4 nm and 340 nm for the TEM-1-CAZ, M184-CAZ, M203-CAZ and M210-CAZ systems respectively suggested that an equilibrium was established between the respective two species and reflected the formation of complexes between them. Therefore, these observations signify that the ground state complexes are formed between respective wild type and mutant βlactamase proteins and CAZ. The UVVis absorption of the four βlactamases increased and a slight redshift that occurred with an increase in concentration of CAZ, which indicated that, the drug-protein interaction resulted in subtle conformational changes in the protein in presence of the drug. A hyper- chromic effect and slight redshift of the maximum absorption peak were also separately observed with increasing CAZ concentrations in solution. The shift of maximum wavelength signifies hydrophobic effect on Trp residue due to complex formation between drug and respective proteins [24]. However, the observations obtained from absorption studies were not sufficient to study the interactions in detail. Therefore, other excited state spectroscopic techniques e.g., fluorescence spectroscopy, TCSPC etc. were preferred to study the binding mode of protein-drug interactions. Variation in temperature produced a local change in the protein microenvironment as heat disrupted the hydrogen bonds, non-polar hydrophobic interactions, which might produce a local change in the Tryptophan microenvironment and affected the spectroscopic properties of the protein. In this study, the increase of quantum yield of wild type and mutant βlactamases with the ascending temperatures represented the change in the local hydrophobicity in presence of CAZ. Moreover, a decrease in fluorescence intensity (quenching) of the βlactamases (wild type and mutants) with increase in temperature in presence of CAZ, also indicated formation of a new complex which corroborated with the change in the protein microenvironment [ 24 – 2 7]. Additionally, in CAZ-β lactamases system the quenching mechanism might also be either due to simultaneous occurrence of static (ground state) and dynamic (excited state) interaction modes. The static quenching had already been established by ground state complex formation. However, the decrease in fluorescence lifetime of wild type and mutant β lactamases with simultaneous addition of CAZ accounted only for dynamic quenching supported by the linear Stern-Volmer plot. Moreover, the positive deviation of Stern-Volmer plot for CAZ-βlactamase systems demonstrated the simultaneous occurrence of static and dynamic quenching. Furthermore, the Stern-Volmer plots of the wild type β-lactamase in presence of increasing concentration of CAZ at different temperatures indicated a gradual shift towards dynamic mode but for mutant-CAZ complexes a sharp shift towards dynamicity indicated a more rigid interaction of the drug to the mutant enzymes[ 21 , 27 ]. Determination of binding constant and binding stoichiometry of βlactamases in presence of CAZ provided evidence for both static and dynamic quenching. The thermodynamic parameters of binding, i.e., changes in standard enthalpy (∆H 0 ), entropy (∆S 0 ) and Gibb's free energy (∆G 0 ) using van't Hoff isotherm exhibited the nature of interacting forces between drug and protein. Usually, four types of forces play an important role in drug–protein interaction, like electrostatic forces, hydrophobic forces, van der Waals interactions and hydrogen bonding [28]. In this study it was found that the interaction of CAZ with the mutant proteins was predominantly hydrophobic, however van der Waal's interactions and hydrogen bonding was the primary interacting forces in the binding of CAZ to the wild type enzyme. Furthermore, the anisotropy changes demonstrated flexible interactions of the drug with the mutants than the wild type protein which also supported predominance of hydrophobic interaction between the drug and mutant enzymes [29]. The circular dichroism (CD) is a sensitive technique to monitor the conformational changes in proteins upon interaction with ligand molecules [ 18 ]. CD spectra of the wild type and mutant proteins in presence of CAZ further corroborated conformational changes in the secondary as well as tertiary structure alterations in the protein at pH 7.0. Additionally, assessment of energy transfer efficiency values (E) using FRET technique and Raman spectroscopy provided evidence that the specific mutations in the M184, M203 and M210 assisted an effective binding of CAZ to the mutant β lactamases than to the wild type protein [ 20 , 30 , 31 ]. In silico MD simulation analysis of the wild type and the mutant β lactamases revealed increase in the RMSD for the C-α backbone for the mutant β lactamases which might be attributed to the overall conformational flexibility of the protein to accommodate the drug molecule [ 21 , 32 ]. Therefore, the overall comparison of RMSD plots for wild type and the variants showed that the C-α backbone residues in the latter maintained stabilizing interaction with the drug molecule. Similarly, RMSF for Cα side chain of the mutant proteins (M184, M203, M210) compared to wild type in presence of the drug showed similar pattern as the RMSF for Cα backbone which further documented the enhanced conformational flexibility in the mutant β-lactamases to bind to CAZ than the wild type [ 14 , 33 ]. The Cα Radius of gyration (Rg) for compactness in the mutantβ lactamases in presence of CAZ also demonstrated a tight binding of the drug to the mutant proteins in comparison to wild type [ 14 , 34 ]. Furthermore, the residue-based conformational changes in the mutants in presence of CAZ also indicated that the altered secondary structure elements (SSE) in the mutants probably imparted additional overall stability and rendered conformational flexibility compared to the wild type protein [ 33 ]. This in-silico study established that the mutant-CAZ interactions were more stable and rigid with minimum conformational alterations between the interacting residues compared to the wild type-CAZ interaction [ 35 ]. Conclusion This is the first study of its kind that illustrated the role of far-site mutations in clinical TEM β-lactamase variants that facilitated ceftazidime (CAZ) binding to the active site of the mutant proteins thus resulting in the inactivation of the drug. In this study, we demonstrated that the far-site mutations favored rigid, mostly hydrophobic interactions with CAZ, as opposed to hydrogen-bond-dominated binding of the drug with the wild-type enzyme by biophysical approach. In silico study also revealed that the mutations conferred conformational flexibility, enabling more firm and stable accommodation of CAZ within the enzyme's active site. Therefore, widespread rampant therapeutic application of β-lactams likely selects and propagates such far-site variant β-lactamases as a survival alternative under therapeutic pressures necessitating new strategies to be developed in drug designing to inhibit mutational adaptations outside the traditional active-site of the mutant proteins. Declarations Funding This research work was supported by an extramural grant from the Department of Biotechnology, Government of India (BT/267/NE/TBP/2011 dated 24.11.2014). Competing Interests The authors declare no competing interests. Author Contributions SKM, MM conceptualized and designed the study. Material preparation, data collection and methodology of the study was performed by SKM. SKM, PPM, MM analyzed the data. The original draft was written by SKM. Final review and editing were done by PPM and MM. Overall supervision and funding acquisition was done by MM. All authors read and agreed to the final version of the manuscript. Data Availability This manuscript does not report data generation or analysis. Acknowledgments This work was carried out at Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, Kolkata and Saha Institute of Nuclear Physics (SINP), Kolkata, West Bengal, India, supported by an extramural grant from the Department of Biotechnology, Government of India (BT/267/NE/TBP/2011 dated 24.11.2014). The authors express their gratitude to the Director, SINP, Kolkata and the Director, School of Tropical Medicine, Kolkata, West Bengal, India, for their support towards the successful completion of the research work. References Avci FG, Altinisik FE, Vardar Ulu D, Ozkirimli Olmez E, Sariyar Akbulut B (2016) An evolutionarily conserved allosteric site modulates beta-lactamase activity. J Enzyme Inhib Med Chem 31:33-40. https://doi.org/10.1080/14756366.2016.1201813 Nwobodo DC, Ugwu MC, Anie CO, Al-Ouqaili MTS, Ikem JC, Chigozie UV, Saki M (2022) Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. Clin Lab Anal 36. https://doi.org/10.1002/jcla.24655 Díaz N, Sordo TL, Merz KM, Suárez D (2003) Insights into the Acylation Mechanism of Class A β-Lactamases from Molecular Dynamics Simulations of the TEM-1 Enzyme Complexed with Benzylpenicillin. J Am Chem Soc 125:672-684. https://doi.org/10.1021/ja027704o Stojanoski V, Chow DC, Hu L, Sankaran B, Gilbert HF, Prasad BVV, Palzkill T (2015) A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis. J Biol Chem 290:10382-10394. https://doi.org/10.1074/jbc.M114.633438 Singh MK, Dominy BN (2012) The Evolution of Cefotaximase Activity in the TEM β-Lactamase. J Mol Biol 415:205-220. https://doi.org/10.1016/j.jmb.2011.10.041 Kumar D, Singh AK, Ali MR, Chander Y (2014) Antimicrobial Susceptibility Profile of Extended Spectrum β-Lactamase (ESBL) Producing Escherichia coli from Various Clinical Samples. Infect Dis (Auckl) 7:IDRT.S13820. https://doi.org/10.4137/IDRT.S13820 Drawz SM, Bonomo RA (2010) Three Decades of β-Lactamase Inhibitors. Clin Microbiol Rev 23:160-201. https://doi.org/10.1128/CMR.00037-09 Salverda MLM, De Visser JAGM, Barlow M (2010) Natural evolution of TEM-1 β-lactamase: experimental reconstruction and clinical relevance. FEMS Microbiol Rev 34:1015-1036. https://doi.org/10.1111/j.1574-6976.2010.00222.x Pimenta AC, Fernandes R, Moreira IS (2014) Evolution of drug resistance: insight on TEM β-lactamases structure and activity and β-lactam antibiotics. Mini Rev Med Chem 14:111-122. https://doi.org/10.2174/1389557514666140123145809 Tooke CL, Hinchliffe P, Bragginton EC, Colenso CK, Hirvonen VHA, Takebayashi Y, Spencer J (2019) β-Lactamases and β-Lactamase Inhibitors in the 21st Century. J Mol Biol 431:3472-3500. https://doi.org/10.1016/j.jmb.2019.04.002 Cantón R, Novais A, Valverde A, Machado E, Peixe L, Baquero F, Coque TM (2008) Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect 14:144-153. https://doi.org/10.1111/j.1469-0691.2007.01850.x Möller M, Denicola A (2002) Protein tryptophan accessibility studied by fluorescence quenching. Biochem Mol Biol Educ 30:175-178. https://doi.org/10.1002/bmb.2002.494030030035 Kumar V, Lakshman PK, Prasad TK, Manjunath K, Bairy S, Vasu AS, Ganavi B, Jasti S, Kamariah N (2024) Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening. Heliyon 10(1):e23864. https://doi.org/10.1016/j.heliyon.2023.e23864 Mukherjee SK, Mandal RS, Das S, Mukherjee M (2018) Effect of non-β-lactams on stable variants of inhibitor-resistant TEM β-lactamase in uropathogenic Escherichia coli: implication for alternative therapy. J Appl Microbiol 124:667-681. https://doi.org/10.1111/jam.13671 Swain BC, Mukherjee SK, Rout J, Sakshi, Mishra PP, Mukherjee M, Tripathy U (2020) A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine. Anal Bioanal Chem 412:2565-2577. https://doi.org/10.1007/s00216-020-02483-1 Mandal P, Bardhan M, Ganguly T (2012) Spectroscopic investigations to reveal the nature of interactions between the haem protein myoglobin and the dye rhodamine 6G. Luminescence 27:285-291. https://doi.org/10.1002/bio.1348 Mishra PP, Patel S, Datta A (2006) Effect of Increased Hydrophobicity on the Binding of Two Model Amphiphilic Chlorin Drugs for Photodynamic Therapy with Blood Plasma and Its Components. J Phys Chem B 110:21238-21244. https://doi.org/10.1021/jp0615858 Bertucci C, Pistolozzi M, De Simone A (2010) Circular dichroism in drug discovery and development: an abridged review. Anal Bioanal Chem 398:155-166. https://doi.org/10.1007/s00216-010-3959-2 Miani A, Raugei S, Carloni P, Helfand MS (2007) Structure and Raman Spectrum of Clavulanic Acid in Aqueous Solution. J Phys Chem B 111:2621-2630. https://doi.org/10.1021/jp066135u Chakraborty B, Mitra P, Basu S (2015) Spectroscopic exploration of drug-protein interaction: a study highlighting the dependence of the magnetic field effect on inter-radical separation distance formed during photoinduced electron transfer. RSC Adv 5:81533-81545. https://doi.org/10.1039/C5RA13575C Mukherjee SK, Mukherjee M, Mishra PP (2021) Impact of Mutation on the Structural Stability and the Conformational Landscape of Inhibitor-Resistant TEM β-Lactamase: A High-Performance Molecular Dynamics Simulation Study. J Phys Chem B 125:11188-11196. https://doi.org/10.1021/acs.jpcb.1c05988 Abdul Samad F, Suliman BA, Basha SH, Manivasagam T, Essa MM (2016) A Comprehensive In Silico Analysis on the Structural and Functional Impact of SNPs in the Congenital Heart Defects Associated with NKX2-5 Gene - A Molecular Dynamic Simulation Approach. PLoS ONE 11:e0153999. https://doi.org/10.1371/journal.pone.0153999 Mátyus L, Szöllősi J, Jenei A (2006) Steady-state fluorescence quenching applications for studying protein structure and dynamics. J Photochem Photobiol B 83:223-236. https://doi.org/10.1016/j.jphotobiol.2005.12.017 Topală T, Bodoki A, Oprean L, Oprean R (2014) Bovine serum albumin interactions with metal complexes. Med Pharm Rep 87:215-219. https://doi.org/10.15386/cjmed-357 Ghisaidoobe A, Chung S (2014) Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques. Int J Mol Sci 15:22518-22538. https://doi.org/10.3390/ijms151222518 Chib R, Butler S, Raut S, Shah S, Borejdo J, Gryczynski Z, Gryczynski I (2015) Effect of quencher, denaturants, temperature and pH on the fluorescent properties of BSA protected gold nanoclusters. J Luminescence 168:62-68. https://doi.org/10.1016/j.jlumin.2015.07.030 Bardhan M, Chowdhury J, Ganguly T (2011) Investigations on the interactions of aurintricarboxylic acid with bovine serum albumin: Steady state/time resolved spectroscopic and docking studies. J Photochem Photobiol B 102:11-19. https://doi.org/10.1016/j.jphotobiol.2010.08.011 Ross PD, Subramanian S (1981) Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 20:3096-3102. https://doi.org/10.1021/bi00514a017 Zhao Q, Tao J, Uppal JS, Peng H, Wang H, Le XC (2019) Nucleic acid aptamers improving fluorescence anisotropy and fluorescence polarization assays for small molecules. TrAC Trends Anal Chem 110:401-409. https://doi.org/10.1016/j.trac.2018.11.018 Lee MM, Peterson BR (2016) Quantification of small molecule-protein interactions using FRET between tryptophan and the pacific blue fluorophore. ACS Omega 1:1266-1276. https://doi.org/10.1021/acsomega.6b00356 Pezzotti G (2021) Raman spectroscopy in cell biology and microbiology. J Raman Spectrosc 52:2348-2443. https://doi.org/10.1002/jrs.6204 Gill M, McCully ME (2019) Molecular dynamics simulations suggest stabilizing mutations in a de novo designed α/β protein. Protein Eng Des Sel 32:317-329. https://doi.org/10.1093/protein/gzaa005 Fantini S, Lisi S, De Los Rios P, Cattaneo A, Pastore A (2020) Protein Structural Information and Evolutionary Landscape by In Vitro Evolution. Mol Biol Evol 37:1179-1192. https://doi.org/10.1093/molbev/msz256 Galdadas I, Qu S, Oliveira ASF, Olehnovics E, Mack AR, Mojica MF, Agarwal PK, Tooke CL, Gervasio FL, Spencer J, Bonomo RA, Mulholland AJ, Haider S (2021) Allosteric communication in class A β-lactamases occurs via cooperative coupling of loop dynamics. ELife 10:e66567. https://doi.org/10.7554/eLife.66567 Yang J, Naik N, Patel JS, Wylie CS, Gu W, Huang J, Ytreberg FM, Naik MT, Weinreich DM, Rubenstein BM (2020) Predicting the viability of beta-lactamase: How folding and binding free energies correlate with beta-lactamase fitness. PLoS ONE 15:e0233509. https://doi.org/10.1371/journal.pone.0233509 Tables Table 1 To 3 are available in the Supplementary Files section. Table 4 Variation in a-helix% of TEM-1(wild type protein) and mutant β‑lactamases (M184, M203 and M210) with increase concentration of Ceftazidime (CAZ). [β‑lactamases]M [CAZ]M α – helix (%) TEM-1(~5x10 -6 M) 0 59.24 ~0.5 x10 -5 M 41.00 ~1.0 x10 -5 M 27.17 ~1.5 x10 -5 M 26.72 ~2.0 x10 -5 M 20.72 M184(~5x10 -6 M) 0 44.10 ~0.5x10 -5 M 42.55 ~1.0 x10 -5 M 34.17 ~1.5 x10 -5 M 33.27 ~2.0 x10 -5 M 14.06 M203(~5x10 -6 M) 0 69.44 ~0.5 x10 -5 M 55.75 ~1.0 x10 -5 M 49.51 ~1.5 x10 -5 M 30.06 ~2.0 x10 -5 M 10.34 M210(~5x10 -6 M) 0 79.10 ~0.5 x10 -5 M 76.75 ~1.0 x10 -5 M 53.31 ~1.5 x10 -5 M 40.72 ~2.0 x10 -5 M 33.68 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.pdf Table1To3.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":1022563,"visible":true,"origin":"","legend":"\u003cp\u003eSteady-state UV–vis absorption spectra and fluorescence emission spectra of TEM-β-lactamase (A,E) and mutant β-lactamases 184M (B,F), M203 (C,G), M210 (D,H) at 5 µM concentration of the respective protein in solution in presence of increasing concentration of CAZ (0-20 µM ). Inset in (A) represented steady-state UV–vis absorption spectra of CAZ at 20 µM at 298 K.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/4ffaf52cd5067022befa7a7c.jpg"},{"id":92616372,"identity":"3b681373-a2fe-4e6b-b97b-838d36c30d9e","added_by":"auto","created_at":"2025-10-01 17:39:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":807178,"visible":true,"origin":"","legend":"\u003cp\u003eStern-Volmer plot and Modified Stern-Volmer plot of Fluorescence quenching of TEM-β lactamase (wild type protein) and mutant β lactamases (M184, M203, M210) at 5 µM concentration in the presence of CAZ (0-20 µM) at 298 K (A,E), 303K (B,F), 308K (C,G) and 313K (D,H). Nonlinear fitting analysis was performed by plotting F0/F against [Q] and Log F0-F/F1 against Log{Q} respectively.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/7ae360bf28a2708fccab7e02.jpg"},{"id":92618535,"identity":"044903e3-0b5f-47fb-8fe7-ed2795d92e89","added_by":"auto","created_at":"2025-10-01 18:11:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":427600,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence lifetime (τ) of TEM-β lactamase (wild type protein) in comparison with mutant β lactamases (M184, M203 and M210) at 5 µM concentration in the presence of CAZ (0-20 µM) at temperature 298K (A) and Fluorescence anisotropy (r) plot of CAZ at 5 µM concentration in presence of increasing concentrations (5-20 µM) of the β lactamases (TEM 1, M184, M203, M210) (B).\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/2b6ee67d4e6014410abab038.jpg"},{"id":92617305,"identity":"e1e83165-1879-4827-a8f8-a59ab10f1152","added_by":"auto","created_at":"2025-10-01 17:47:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":518034,"visible":true,"origin":"","legend":"\u003cp\u003eVan't Hoff plot for the binding of TEM-β lactamases (wild type protein) (A) in comparison with mutant β lactamases M184(B), M203(C), M210(D) at 5 µM concentration in the presence of CAZ (0-20µM) concentration at different temperatures 298K, 303K, 308K and 311K.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/eb6385e5095d42e33d278dca.jpg"},{"id":92618097,"identity":"257bb4ef-4a2d-4694-8258-9b031c569cc2","added_by":"auto","created_at":"2025-10-01 18:03:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1004744,"visible":true,"origin":"","legend":"\u003cp\u003eCα backbone RMSD, Cα all residue RMSD, RMSF for Cα backbone, RMSF for Cα side chain and Rg for backbone Cα for 100 ns simulation time frame shown for the wild type TEM-1 (1ZG4) interacting with CAZ; displayed in black, in comparison with mutant proteins M184 (A,D,G,J,M), M203 (B,E,H,K,N), and M210 (C,F,I,L,O); displayed in red respectively. Projection of SSEs throughout the MD simulation trajectory in presence of CAZ for wild type TEM-1 (IZG4) (P), M184 (Q), M203 (R), M210 (S). SSE composition is represented by different color codes: helices; pink, turns; green, 3-10 helix; blue, β-strands; yellow, coils; white.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/0f93714407dec17f008d74db.jpg"},{"id":93876145,"identity":"7d4975b4-3615-4bb3-8368-1a60d6051c15","added_by":"auto","created_at":"2025-10-19 14:08:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4694111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/060b44b4-9ec7-40ae-94d2-1efa9c3217eb.pdf"},{"id":92616379,"identity":"a7a4b63d-1a5f-43ee-b8e8-07583672c8cc","added_by":"auto","created_at":"2025-10-01 17:39:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":597609,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/7f6997e4375f38fc6ac1f65a.pdf"},{"id":92616370,"identity":"d15a9e35-02c0-44d9-b4c4-e2223b74e3fc","added_by":"auto","created_at":"2025-10-01 17:39:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27561,"visible":true,"origin":"","legend":"","description":"","filename":"Table1To3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7504842/v1/e576ea63e946f3450e401b47.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Distant site mutations in clinical TEM β-lactamase variants enhance non-covalent binding to ceftazidime: Insights from biophysical and in silico investigations","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePersistence of bacterial resistance against antimicrobial agents is a global health threat [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Production of active site serine β-lactamase enzymes is the major mechanism of antibiotic resistant in uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (UPEC) that catalyze the hydrolysis of β-lactam antibiotics. [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Currently, based on Ambler classification scheme, β-lactamases can be grouped into four classes A, B, C and D. Class A, C, and D are serine β-lactamases (non-metallo) that utilize the serine residue to catalyze the hydrolysis of antibiotics, whereas class B enzymes are metallo-β-lactamases and need zinc ions in the active site to attack the β-lactam ring [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. .In the case of Class A β-lactamases which is prime concerned for the clinicians due to its wide range of hydrolyzing activity against antibiotics mostly against β-lactam antibiotics where activation of catalytic Ser70 conserved residue initiates nucleophilic attack on the β-lactam carbonyl carbon and opening of the β-lactam ring to form a covalently linked acyl-enzyme intermediate and ultimately the attack leads to deacylation of the acyl-enzyme species resulting inactivation of antibiotic and regeneration of the free enzyme [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].TEM1 β-lactamase was identified as one of the most evolved Class A β-lactamases in hospital infection, mediated by plasmid found in \u003cem\u003eE. coli\u003c/em\u003e resistant to antibiotics and also considered as the precursor of the TEM enzyme family with three major types of activity; the broad-spectrum β-lactamases, the extended spectrum β-lactamases (ESBLs) and the inhibitor resistant β-lactamases. The emergence of resistance mediated by this TEM enzyme family were most explored β-lactamases till date and it was found that their extensive structural adjustment by their sustainable mutational modifications led to the generation of distinct multiple variants with in-depth structural diversity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To overcome β-lactam resistance extensive studies were carried out against the acylation mechanism and two types of β-lactams were introduced such as extended spectrum cephalosporins; and β-lactamase inhibitors, such as clavulanic acid, sulbactam and tazobactam [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, the structural alterations in Class A β-lactamases impair the antibacterial activities of the inhibitors by covalent interaction between the active site residues in the former with the inhibitory molecules resulting in resistance against them [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Till date\u0026thinsp;~\u0026thinsp;200 TEM-variants have been reported that confers resistance to novel β-lactams and their inhibitor combinations which were introduced in the past three decades [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Studies from the different part of the world showed that the indiscriminate use of β-lactam-β-lactamase inhibitor combination has increased the number of TEM β-lactamase variants; the extended-spectrum (ES) and inhibitor-resistant phenotypes (IR) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous reports showed that mutation or substitution in the active site Ω-loop region of TEM β-lactamase not only increased the resistance phenotype against cephalosporins but also increased the structural flexibility imparting enlargement of the active site pocket loop that can accommodate newer β-lactams irrespective of their -R group thus decreasing the activity of oxyimino-cephalosporin, ceftazidime (CAZ), and all other β-lactam-β-lactamase inhibitors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Fluorescence techniques were widely used as one of the important biophysical application that paved sensitivity, reliability and rapidity in the field of drug-protein interactions study [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThere are not many Indian studies that described existence and characterization of TEM β-lactamase mutants that harbor mutations at sites other than the active site of the enzyme isolated from clinical origin as a result of random drug administration. An earlier study from our laboratory, demonstrated identification and characterization of three novel TEM β-lactamase mutants; pUE184TEM (M184), pUE203TEM (M203), pUE210TEM (M210) from clinical uropathogenic \u003cem\u003eE. coli\u003c/em\u003e isolates with mutation at residues other than those that constituted the active site of the enzymes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the present study bioactive interactions of CAZ with the mutant β-lactamases under physiological conditions was investigated. The role of distant site mutations on the conformational flexibility in the mutants to accommodate CAZ was explored by using a collection of biophysical and \u003cem\u003ein silico\u003c/em\u003e methods to obtain an insight into selection and dissemination of the resistant β-lactamases in response to uncontrolled β-lactam administration.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAbsorbance Spectroscopy\u003c/h2\u003e\u003cp\u003eAbsorbance spectral measurement was recorded in UV\u0026ndash;vis Spectrometer at a scan rate of 480 nm/min with 1 cm-path-length quartz cuvette in the visible wavelength range of 200-800nmat different temperatures (298K, 303K, 308K, 311K).The titrations of the experiment were conducted on 5 \u0026micro;M wild type TEM βlactamase and its variants (M184, M203, M210) [14)] in presence of ascending concentration of CAZ (5 \u0026micro;M-20\u0026micro;M) at respective temperatures. The inner filter effect was eliminated using the relationship; I\u003csub\u003ecor\u003c/sub\u003e = I\u003csub\u003eobs\u003c/sub\u003ee\u003csup\u003e(Aex+Aem)/2\u003c/sup\u003e ,where the corrected fluorescence intensity is denoted as I\u003csub\u003ecorr\u003c/sub\u003e, and observed fluorescence intensity is denoted as I\u003csub\u003eobs\u003c/sub\u003e. The A\u003csub\u003eex\u003c/sub\u003e and A\u003csub\u003eem\u003c/sub\u003e denote absorbance values at excitation and emission wavelengths, respectively [15].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFluorescence Spectroscopy\u003c/h3\u003e\n\u003cp\u003eFluorescence emission spectrum was recorded in the range of 280\u0026ndash;600 nm using Hitachi F-7000 Spectrofluorimeter with excitation at 295 nm. Both the excitation and emission bandwidth were fixed at 5 nm. Quenching experiment was carried out in presence of increasing concentration of CAZ (5 \u0026micro;M-20\u0026micro;M) solution at different temperatures (298K, 303K, 308K, 311K) to 5 \u0026micro;M concentration of the wild type and the mutant βlactamases. Static fluorescence quenching was determined by using the Stern\u0026ndash;Volmer (SV) equation from steady state measurements F\u003csub\u003e0\u003c/sub\u003e/F\u0026thinsp;=\u0026thinsp;1\u0026thinsp;+\u0026thinsp;K\u003csub\u003esv\u003c/sub\u003e(Q), where F\u003csub\u003e0\u003c/sub\u003e and F denote the steady-state fluorescence emission intensities in the absence and in presence of CAZ. K\u003csub\u003eSV\u003c/sub\u003e is the Stern\u0026ndash;Volmer constant and (Q) is the concentration of the quencher. Collisional or dynamic fluorescence quenching was determined using the modified SternVolmer equation; log ((F\u003csub\u003e0\u003c/sub\u003e -F)/F))\u0026thinsp;=\u0026thinsp;logK\u003csub\u003ea\u003c/sub\u003e + n log (Q), where K\u003csub\u003ea\u003c/sub\u003e denoted the binding constant and n the number of binding sites.\u003c/p\u003e\n\u003ch3\u003eFluorescence lifetime measurements\u003c/h3\u003e\n\u003cp\u003eThe fluorescence lifetime(τ) measurements determine energy transfer at the molecular level between interacting species. Time correlated single photon counting (TCSPC) was used to measure the fluorescence life times of wild type and mutant βlactamases in gradually increasing concentration of CAZ (0 \u0026micro;M-20\u0026micro;M) in sodium phosphate buffer (pH 7.4) at 298K [16].\u003c/p\u003e\n\u003ch3\u003eQuantum yield and fluorescence anisotropy\u003c/h3\u003e\n\u003cp\u003eThe quantum yield (Φ) or quantum efficiency of CAZ at 5\u0026micro;M concentration in sodium phosphate buffer (pH 7.4) was recorded by monitoring the fluorescence emission intensity in presence of increasing concentrations of the wild type and mutant β lactamases (0\u0026ndash;20 \u0026micro;M)at 298K [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The fluorescence anisotropy (r) measurement of CAZ at 5\u0026micro;M concentration in 5mM sodium phosphate buffer (pH 7.4) was recorded against addition of ascending concentration of wild type and mutant proteins (5\u0026micro;M-20\u0026micro;M) in Fluromax Spectrofluorometer. All measurements were conducted at 298K at 3 minutes of interval.\u003c/p\u003e\n\u003ch3\u003eThermodynamic properties\u003c/h3\u003e\n\u003cp\u003eVan't Hoff isotherm equation was used to determine the thermodynamic parameter (kinetic and equilibrium properties) that govern the interaction between the ligands (CAZ) and the proteins (TEM and its mutants). The values of ∆H\u003csup\u003e0\u003c/sup\u003e and ∆S\u003csup\u003e0\u003c/sup\u003e were obtained from the \u003cem\u003elnK\u003c/em\u003e vs. 1/T plot, where ∆G\u003csup\u003e0\u003c/sup\u003e, ∆H\u003csup\u003e0\u003c/sup\u003e, and ∆S\u003csup\u003e0\u003c/sup\u003e are depicted as standard enthalpy change, standard free energy change, and standard entropy change respectively.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCircular Dichroism (CD) Spectroscopy\u003c/h2\u003e\u003cp\u003eFar and Near UV-CD spectra was recorded at room temperature for βlactamases (TEM and its mutants) in presence of ascending concentration of ceftazidime ranges from 0 \u0026micro;M-20\u0026micro;M solutions using JASCO-J815 spectrometer with a temperature controller at 298 K in a quartz cuvette of 1 mm path length. Each final spectrum was recorded as an average over three scans. The far and near-UV-CD region was recorded as 250\u0026ndash;360 nm and 360 nm to 500 nm.CD results were expressed as mean residue elasticity (MRE) in degree cm\u003csup\u003e2\u003c/sup\u003e mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The secondary structures (α-helix, β-sheet, random coils) were calculated from respective MRE values [18].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRaman spectroscopy\u003c/h3\u003e\n\u003cp\u003eRaman scattering was recorded for βlactamases (TEM and its mutant variants) in presence of ascending concentration of ceftazidime ranges from 0\u0026micro;M-20\u0026micro;M. The spectral range was taken for500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with an integration time of 20 s using a 670 nm red laser excitation, at 10 mW power [19].\u003c/p\u003e\n\u003ch3\u003eFörster resonance energy transfer (FRET) computation\u003c/h3\u003e\n\u003cp\u003eFRET involves a distance dependent physical method of non-radiative transfer of energy between an excited fluorophore (donor) and another fluorophore (acceptor) by intermolecular dipole-dipole interactions. The energy transfer efficiency (E) signified the fraction of photons absorbed by the donor moiety which was transferred to the acceptor. E\u0026thinsp;=\u0026thinsp;1-(F/F\u003csub\u003e0\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;R\u003csub\u003e0\u003c/sub\u003e\u003csup\u003e6\u003c/sup\u003e/(R\u003csub\u003e0\u003c/sub\u003e\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;r\u003csup\u003e6\u003c/sup\u003e), where, r represented the distance between the donor and acceptor and R\u003csub\u003e0\u003c/sub\u003e represented the critical energy transfer distance, at which 50% of the excitation energy was transmitted from the former to the latter. R\u003csub\u003e0\u003c/sub\u003e was evaluated from the following equation; R\u003csub\u003e0\u003c/sub\u003e\u003csup\u003e6\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;8.79 x 10\u003csup\u003e\u0026minus;\u0026thinsp;25\u003c/sup\u003eK\u003csup\u003e2\u003c/sup\u003eN\u003csup\u003e\u0026minus;4\u003c/sup\u003eϕ, where K\u003csup\u003e2\u003c/sup\u003e was the spatial orientation factor of the altered dipoles of the acceptor and the donor, N represented the refractive index of the medium, ϕ denoted the fluorescence quantum yield of the donor in absence of the acceptor [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMolecular dynamics simulation\u003c/h2\u003e\u003cp\u003eMolecular dynamics and simulation were performed on the TEM β-lactamase (1ZG4) and its variants [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] in presence of CAZ to determine the binding stability of the protein\u0026ndash;drug docked complex using Desmond v3.6 Package. Topology of the protein molecules were generated by optimized potentials for liquid simulation (OPLS) force field. Simple point charge (SPC) water model was used to perform the protein\u0026ndash;drug complexes Simulation with a distance of 10 \u0026Aring; between the complex and the periodic box. System was neutralized by adding Na+/Cl\u0026thinsp;\u0026minus;\u0026thinsp;ions and placed randomly in the solvated system. To minimize and relax the protein/protein\u0026thinsp;\u0026minus;\u0026thinsp;drug complex under NPT ensemble, the default protocol of Desmond was used followed by total of nine stages among which two minimization and four short simulation (equilibration phase) steps was involved before starting the actual production time. System temperature and pressure was 300 K and 1 atmosphere, respectively, using Nose\u0026thinsp;\u0026minus;\u0026thinsp;Hoover temperature coupling and isotropic scaling, and the simulation was performed for 100 ns NPT production simulation and saving the configurations at 10 ns intervals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSimulation trajectory analysis\u003c/h2\u003e\u003cp\u003eSimulation trajectory files were analyzed using Desmond module programs i.e. simulation quality analysis (SQA), simulation event analysis (SEA), and simulation interaction diagram (SID) to calculate energies, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), the radius of gyration (Rg) and the secondary structure elements (SSEs) which pave the protein structure stability. SQA was used to qualitatively validate the system stability throughout the simulated length of chemical time for the given temperature, pressure, and volume of the total simulation box, SEA was used to analyze each frame of the simulated trajectory output, whereas total SSE change in the protein structure in presence of the drug during the simulation time was estimated using SID [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eSpectroscopic measurements\u003c/h2\u003e\n \u003cp\u003eUVVis absorption spectra of wild type and mutant βlactamases (Fig.\u0026nbsp;1A-D) were recorded in presence of increasing concentrations of ceftazidime, at 298K. All protein showed a broad band at 280 nm while ceftazidime had a strong absorbance at 267 nm and a moderate absorbance at 310 nm. With gradual increase of CAZ concentration, the absorbance spectra of wild βlactamase showed enhancement of absorption intensity.\u003c/p\u003e\n \u003cp\u003eThe possibility of ground state complex formation couldnot be ruled out from the above absorption spectra. Additionally, from Fig. S1 it was observed that the spectra of the mixture of CAZ and wild type βlactamase (\u003cstrong\u003ecurve 5\u003c/strong\u003e) was much intense than intensity of spectra corresponding to the summation of the two absorption bands of the individual reacting components (\u003cstrong\u003ecurve 3\u003c/strong\u003e). The \u003cstrong\u003ecurve 4\u003c/strong\u003e obtained by subtracting the \u003cstrong\u003ecurve 3\u003c/strong\u003e from the \u003cstrong\u003ecurve 5\u003c/strong\u003e indicated formation of ground state complex between the two species.\u003c/p\u003e\n \u003cp\u003eThe UV-Vis absorption spectra of the wild type and mutant β lactamases in presence of CAZ were recorded at 303 K, 308 K and 311 K respectively which indicated that the proteins-CAZ complexes were thermally stable (\u003cstrong\u003eFig. S2\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eThe fluorescence spectra of wild type and mutant βlactamases at pH 7.5in the presence of 5, 10, 15, 20µM CAZ at 298K showed strong fluorescence intensity at 340 nm upon excitation at 280 nm which gradually decreased upon addition of CAZ with a slight red shift at 298 K (Fig.\u0026nbsp;1E-H, \u003cstrong\u003eFig. S3\u003c/strong\u003e). Similar comparable fluorescence spectra of the βlactamases at the different temperatures (303K.308K, 311K) were also recorded (data not shown).\u003c/p\u003e\n \u003cp\u003eThe quenching phenomenon exhibited in the fluorescence emission spectra of the proteins and protein –CAZ complexes were analyzed by Stern–Volmer (SV) plot. The non-linearity observed in the SV plot at 298K, 303K, 308K, 311K (Fig. 2A-D) indicated a combination of static (ground state) and dynamic (excited state) quenching which was validated by modified SV plots (Fig. 2E-H). Additionally, SV plots of quenching of the wild type in presence of increased concentration of CAZ at different temperatures indicated a gradual shift towards dynamic mode although mutants exhibited a decrease in their dynamic mode of quenching.\u003c/p\u003e\n \u003cp\u003eFurthermore, fluorescence lifetime changes of the wild type and the mutant proteins in presence of different concentrations of CAZ also exhibited a steady decrease (Fig. 3A, Table 1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eFluorescence quantum yield and fluorescence anisotropy of CAZ\u003c/h2\u003e\n \u003cp\u003eAbsolute fluorescence quantum yield was also analyzed for small molecule i.e. CAZ to measure changes in radiative rates from changes in nonradiative rates occurring in terms of interaction. The quantum yields of the CAZ molecule increased with the increase in concentration of proteins (wild and all three mutant βlactamases). The quantum yields of CAZ increased gradually from 0.08 to 0.27, 0.36, 0.36 and 0.37 for the wild type, M184, M203 and M210 βlactamase respectively (Table 2).Fluorescence anisotropy was also performed to measure the microenvironment around CAZ in terms of rotational diffusion and interactions. The anisotropy of the CAZ molecule was very low (r = 0.07). With increased concentration of the proteins (wild type and all three mutant βlactamases), fluorescence anisotropy of CAZ increased gradually. At 20µM concentration of CAZ a steady increase in r value from 0.18, 0.20, 0.21 to 0.24 in presence of the respective βlactamases (wild type, M184, M203, M210) respectively (Fig. 3B)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eBinding constants, number of binding sites and mode of binding\u003c/h2\u003e\n \u003cp\u003eThe steady state fluorescence data were further evaluated by the binding affinity (K\u003csub\u003eb\u003c/sub\u003e) of CAZ molecule to the equivalent sites of wild type and mutant βlactamases independently. For mutant βlactamases a progressive decrease of binding affinity (K\u003csub\u003eb\u003c/sub\u003e) was observed with the increase of temperature whereas in case of wild type a vice versa affinity was observed in pH 7.5.(Fig. 4).\u003c/p\u003e\n \u003cp\u003eThe equilibrium interaction (such as, hydrogen bonds, van der Waals forces, electrostatic forces, and/or hydrophobic associations), between ligand (CAZ) and protein molecule were determined from the value of the thermodynamic parameter (∆G\u003csup\u003e0\u003c/sup\u003e, ∆H\u003csup\u003e0\u003c/sup\u003e, ∆S\u003csup\u003e0\u003c/sup\u003e)using van't Hoff isotherm equation from the lnK vs. 1/T plot. Significant negative ∆G\u003csup\u003e0\u003c/sup\u003evalue, ∆H\u003csup\u003e0\u003c/sup\u003e \u0026lt; 0 and ∆S\u003csup\u003e0\u003c/sup\u003e \u0026gt; 0 indicated spontaneous interaction between the reacting components. Increase in negative ∆G\u003csup\u003e0\u003c/sup\u003e value with increase in positive ∆H\u003csup\u003e0\u003c/sup\u003e and ∆S\u003csup\u003e0\u003c/sup\u003e values in the mutantβlactamases indicated discrete predominance of hydrophobic interaction in contrary to the wild type βlactamase that exhibited van der Waals and H-bond interaction with high negative ∆H\u003csup\u003e0\u003c/sup\u003eand ∆S\u003csup\u003e0\u003c/sup\u003e values compared to less negative ∆G\u003csup\u003e0\u003c/sup\u003e value (Fig. 4, Table 3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eCircular Dichroism (CD) Spectroscopy\u003c/h2\u003e\n \u003cp\u003eConformational changes of wild and mutant βlactamases were further measured by CD spectroscopy in the absence and presence of CAZ at pH 7.5. Results showed that the mean residue ellipticity (MRE) of pure α-helix percentage of wild and mutant βlactamases were decreased upon binding of CAZ demonstrated secondary structure alternations of the proteins in far-UV-CD region (185–250 nm) with two characteristic dips at 209 nm and 222 nm for all the proteins respectively. Moreover, in the near-UV-CD region spectra (240–320 nm) of the wild type and mutant proteins showed comparable minor alterations of their tertiary structure (\u003cstrong\u003eFig. S4\u003c/strong\u003e, Table 4)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eRaman spectrum of βlactamase proteins\u003c/h2\u003e\n \u003cp\u003eRaman intensity of wild type and mutant βlactamases (5µM) were measured in presence of ascending concentration of CAZ (5 µM-15 µM). Results showed a gradual increase of Raman intensity of βlactamases with the increase of the concentration of CAZ in comparison with the wild type protein. There are three regions (880 cm\u003csup\u003e− 1\u003c/sup\u003e, the region of Tyr doublet due to H-Bonding),(1360 cm\u003csup\u003e− 1\u003c/sup\u003e, CH2- CH2 deformation) and (1640–1680 cm\u003csup\u003e− 1\u003c/sup\u003e, characteristics of amide peak) in the spectrum which indicated the secondary structure alternation due to molecular interaction. A small blue shift was also observed for the mutant β lactamases in comparison with the wild type due to secondary structure adjustment during interaction with CAZ. (\u003cstrong\u003eFig. S5\u003c/strong\u003e)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eEnergy transfer between CAZ and β lactamases\u003c/h2\u003e\n \u003cp\u003eThe absorbance spectrum of CAZ (excited at 267 nm) and fluorescence spectrum of the TEM 1 β- lactamase at 340 nm overlapped with each other which satisfied the requisite condition of FRET between the CAZ and the proteins \u003cstrong\u003e(Fig. S6)\u003c/strong\u003e. Moreover, from the Förster theory of energy transfer, the molecular interactions between the drug-protein complexes were evidenced from discrete values of the energy transfer efficiency (E); 0.47, 0.94, 0.79 and 0.82 for CAZ-wild type, CAZ-M184, CAZ-M203 and CAZ-M210 respectively which facilitated binding of CAZ to the mutants than the wild type.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIn silico\u003c/strong\u003e \u003cstrong\u003estudy\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eEarlier \u003cem\u003ein silico\u003c/em\u003e study from our laboratory indicated effective binding of CAZ to the wild type and mutant β lactamases (Mukherjee S K et al, 2018). The stability and conformational changes of the β lactamase in presence of CAZ were analyzed by MD simulation study. The movement of atoms and molecules for 100 ns of simulation were monitored. In presence of CAZ, the wild type β lactamases (1ZG4) and M184 exhibited C-α backbone deviations at about ∼1.5 to 1.75 Å and ∼1.75 to 2.5 Å respectively throughout the simulation trajectory and an equilibrium was maintained after 40 ns for both. However, for M203 and M210 C-α backbone deviations from ∼1.5 to 2.5 and 1.75 to 2.5 Å with an elevation at 60 ns for the latter which reached equilibrium from 70 ns was observed. The C-α all residue deviations for M184 and M210 were ∼2.5 to 3.25 Å and ∼2.5 to 3.40 Å respectively with an elevation at 60 ns and equilibrium from 70ns in case of the latter. In contrast, M203 showed reduced Cα backbone deviation at 30 ns akin to wild type throughout the 100 ns simulation trajectory. However, M203 showed C-α all residue deviations from ∼2.25 to 3.0 in comparison to wild type in presence of CAZ (Fig.\u0026nbsp;5A-F)\u003c/p\u003e\n \u003cp\u003eThe residue level fluctuations (RMSF) of Cα backbone for M184 and M210 in presence of CAZ were moderately high (∼3.25 Å) in comparison to wild type at residues located between 50–80, 120–170, 200, and 225–250 respectively. However, a comparable pattern of fluctuation was observed for M203 and wild type except at residue 150. Similarly, RMSF for Cα side chain of the mutant proteins (M184, M203, M210) in presence of CAZ compared to wild type in presence of the drug showed similar pattern as the RMSF for Cα backbone (Fig.\u0026nbsp;5G-L).\u003c/p\u003e\n \u003cp\u003eThe compactness of protein-drug interaction was ascertained by Cα Radius of gyration (Rg) plot. The residue level Rg value fluctuation for wild type protein was within the range of ∼17.8 to 18.3 Å, whereas for mutant protein (M184) was within the range of ∼17.8 to 18.2 Å and reached equilibrium from 40 ns onward in presence of CAZ. Furthermore, in presence of the drug the Rg value fluctuations for M203 was ∼17.8 to 18.1 across the 100 ns simulation trajectory but for M210, the Rg value fluctuations was ∼17.9 to 18.6 Å with a decrease in fluctuation within the range of ∼18.2 to 18.4 Å from 60 ns onward to attain the equilibrium. The overall Rg analysis for compactness in the mutants in presence of CAZ showed lowest radius of gyration that demonstrated tight binding of CAZ to the mutant proteins in comparison to wild type (Fig.\u0026nbsp;5M-O).\u003c/p\u003e\n \u003cp\u003eThe protein-drug interactions were further monitored by estimation of secondary structure elements (SSE) composition (helices, strands, turns, loops) in the interacting proteins across the simulation trajectory throughout the 100 ns time frame. SSE for each amino acid residue of the mutants exhibited significant structural adjustment in comparison with wild type in presence of CAZ. In M184 increase of 310-helix in residue positions 100, 160, 220, turns in residue position 50, 90, 160, coils in position 140, 200 and π-helix in positions 210 to 225 was evidenced. In M203, the segment of the residues in positions 60–90 showed increase of 310-helix, coil structure at residues 150–170 and 225–240. M210 showed an increase of α-helix and coil with the absence of π-helix at positions 60–90 and 160–170 in comparison with the segment of residues in the wild-type with increase of 310-helix and increase of π-helix at residue position 210–225 respectively (Fig.\u0026nbsp;5P-T).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEmergence of varied β-lactamases was reported which was the primary cause of β-lactam resistance in uropathogenic \u003cem\u003eE. coli\u003c/em\u003e, the primary etiologic agent of urinary tract infection. These β-lactamases were found to harbor mutations which paved the possibility of effective binding of the different β-lactams and their inhibitor combinations to develop resistance. Therefore, studies on the protein-drug interactions have become increasingly important from a pharmaceutical perspective. Absorption spectroscopy study provide information on the binding modes of ligands with proteins pave the possibility of ground state complex formation between protein-ligand interfaces [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The individual UVVis absorption spectrum of the CAZ and TEM-1 βlactamase were markedly different to that of CAZ-βlactamase complex, indicating the formation of new complexes between CAZ and βlactamases. Appearance of isosbestic points at 339.4 nm, 360.2 nm, 341.4 nm and 340 nm for the TEM-1-CAZ, M184-CAZ, M203-CAZ and M210-CAZ systems respectively suggested that an equilibrium was established between the respective two species and reflected the formation of complexes between them. Therefore, these observations signify that the ground state complexes are formed between respective wild type and mutant βlactamase proteins and CAZ. The UVVis absorption of the four βlactamases increased and a slight redshift that occurred with an increase in concentration of CAZ, which indicated that, the drug-protein interaction resulted in subtle conformational changes in the protein in presence of the drug. A hyper- chromic effect and slight redshift of the maximum absorption peak were also separately observed with increasing CAZ concentrations in solution. The shift of maximum wavelength signifies hydrophobic effect on Trp residue due to complex formation between drug and respective proteins [24]. However, the observations obtained from absorption studies were not sufficient to study the interactions in detail.\u003c/p\u003e\u003cp\u003eTherefore, other excited state spectroscopic techniques e.g., fluorescence spectroscopy, TCSPC etc. were preferred to study the binding mode of protein-drug interactions. Variation in temperature produced a local change in the protein microenvironment as heat disrupted the hydrogen bonds, non-polar hydrophobic interactions, which might produce a local change in the Tryptophan microenvironment and affected the spectroscopic properties of the protein. In this study, the increase of quantum yield of wild type and mutant βlactamases with the ascending temperatures represented the change in the local hydrophobicity in presence of CAZ. Moreover, a decrease in fluorescence intensity (quenching) of the βlactamases (wild type and mutants) with increase in temperature in presence of CAZ, also indicated formation of a new complex which corroborated with the change in the protein microenvironment [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e7].\u003c/p\u003e\u003cp\u003eAdditionally, in CAZ-β lactamases system the quenching mechanism might also be either due to simultaneous occurrence of static (ground state) and dynamic (excited state) interaction modes. The static quenching had already been established by ground state complex formation. However, the decrease in fluorescence lifetime of wild type and mutant β lactamases with simultaneous addition of CAZ accounted only for dynamic quenching supported by the linear Stern-Volmer plot. Moreover, the positive deviation of Stern-Volmer plot for CAZ-βlactamase systems demonstrated the simultaneous occurrence of static and dynamic quenching. Furthermore, the Stern-Volmer plots of the wild type β-lactamase in presence of increasing concentration of CAZ at different temperatures indicated a gradual shift towards dynamic mode but for mutant-CAZ complexes a sharp shift towards dynamicity indicated a more rigid interaction of the drug to the mutant enzymes[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDetermination of binding constant and binding stoichiometry of βlactamases in presence of CAZ provided evidence for both static and dynamic quenching. The thermodynamic parameters of binding, i.e., changes in standard enthalpy (∆H\u003csup\u003e0\u003c/sup\u003e), entropy (∆S\u003csup\u003e0\u003c/sup\u003e) and Gibb's free energy (∆G\u003csup\u003e0\u003c/sup\u003e) using van't Hoff isotherm exhibited the nature of interacting forces between drug and protein. Usually, four types of forces play an important role in drug\u0026ndash;protein interaction, like electrostatic forces, hydrophobic forces, van der Waals interactions and hydrogen bonding [28]. In this study it was found that the interaction of CAZ with the mutant proteins was predominantly hydrophobic, however van der Waal's interactions and hydrogen bonding was the primary interacting forces in the binding of CAZ to the wild type enzyme. Furthermore, the anisotropy changes demonstrated flexible interactions of the drug with the mutants than the wild type protein which also supported predominance of hydrophobic interaction between the drug and mutant enzymes [29].\u003c/p\u003e\u003cp\u003eThe circular dichroism (CD) is a sensitive technique to monitor the conformational changes in proteins upon interaction with ligand molecules [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. CD spectra of the wild type and mutant proteins in presence of CAZ further corroborated conformational changes in the secondary as well as tertiary structure alterations in the protein at pH 7.0. Additionally, assessment of energy transfer efficiency values (E) using FRET technique and Raman spectroscopy provided evidence that the specific mutations in the M184, M203 and M210 assisted an effective binding of CAZ to the mutant β lactamases than to the wild type protein [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e MD simulation analysis of the wild type and the mutant β lactamases revealed increase in the RMSD for the C-α backbone for the mutant β lactamases which might be attributed to the overall conformational flexibility of the protein to accommodate the drug molecule [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, the overall comparison of RMSD plots for wild type and the variants showed that the C-α backbone residues in the latter maintained stabilizing interaction with the drug molecule.\u003c/p\u003e\u003cp\u003eSimilarly, RMSF for Cα side chain of the mutant proteins (M184, M203, M210) compared to wild type in presence of the drug showed similar pattern as the RMSF for Cα backbone which further documented the enhanced conformational flexibility in the mutant β-lactamases to bind to CAZ than the wild type [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The Cα Radius of gyration (Rg) for compactness in the mutantβ lactamases in presence of CAZ also demonstrated a tight binding of the drug to the mutant proteins in comparison to wild type [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, the residue-based conformational changes in the mutants in presence of CAZ also indicated that the altered secondary structure elements (SSE) in the mutants probably imparted additional overall stability and rendered conformational flexibility compared to the wild type protein [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This \u003cem\u003ein-silico\u003c/em\u003e study established that the mutant-CAZ interactions were more stable and rigid with minimum conformational alterations between the interacting residues compared to the wild type-CAZ interaction [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is the first study of its kind that illustrated the role of far-site mutations in clinical TEM β-lactamase variants that facilitated ceftazidime (CAZ) binding to the active site of the mutant proteins thus resulting in the inactivation of the drug. In this study, we demonstrated that the far-site mutations favored rigid, mostly hydrophobic interactions with CAZ, as opposed to hydrogen-bond-dominated binding of the drug with the wild-type enzyme by biophysical approach. \u003cem\u003eIn silico\u003c/em\u003e study also revealed that the mutations conferred conformational flexibility, enabling more firm and stable accommodation of CAZ within the enzyme's active site. Therefore, widespread rampant therapeutic application of β-lactams likely selects and propagates such far-site variant β-lactamases as a survival alternative under therapeutic pressures necessitating new strategies to be developed in drug designing to inhibit mutational adaptations outside the traditional active-site of the mutant proteins.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was supported by an extramural grant from the Department of Biotechnology, Government of India (BT/267/NE/TBP/2011 dated 24.11.2014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSKM, MM conceptualized and designed the study. Material preparation, data collection and methodology of the study was performed by SKM. SKM, PPM, MM analyzed the data. The original draft was written by SKM. Final review and editing were done by PPM and MM. Overall supervision and funding acquisition was done by MM. All authors read and agreed to the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript does not report data generation or analysis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out at Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, Kolkata and Saha Institute of Nuclear Physics (SINP), Kolkata, \u0026nbsp;West Bengal, India, \u0026nbsp;supported by an extramural grant from the Department of Biotechnology, Government of India (BT/267/NE/TBP/2011 dated 24.11.2014). The authors express their gratitude to the Director, SINP, Kolkata \u0026nbsp;and the Director, School of Tropical Medicine, Kolkata, West Bengal, India, for their support towards the successful completion of the research work.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAvci FG, Altinisik FE, Vardar Ulu D, Ozkirimli Olmez E, Sariyar Akbulut B (2016) An evolutionarily conserved allosteric site modulates beta-lactamase activity. J Enzyme Inhib Med Chem 31:33-40. https://doi.org/10.1080/14756366.2016.1201813\u003c/li\u003e\n\u003cli\u003eNwobodo DC, Ugwu MC, Anie CO, Al-Ouqaili MTS, Ikem JC, Chigozie UV, Saki M (2022) Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. Clin Lab Anal 36. https://doi.org/10.1002/jcla.24655\u003c/li\u003e\n\u003cli\u003eD\u0026iacute;az N, Sordo TL, Merz KM, Su\u0026aacute;rez D (2003) Insights into the Acylation Mechanism of Class A \u0026beta;-Lactamases from Molecular Dynamics Simulations of the TEM-1 Enzyme Complexed with Benzylpenicillin. J Am Chem Soc 125:672-684. https://doi.org/10.1021/ja027704o\u003c/li\u003e\n\u003cli\u003eStojanoski V, Chow DC, Hu L, Sankaran B, Gilbert HF, Prasad BVV, Palzkill T (2015) A Triple Mutant in the \u0026Omega;-loop of TEM-1 \u0026beta;-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis. J Biol Chem 290:10382-10394. https://doi.org/10.1074/jbc.M114.633438\u003c/li\u003e\n\u003cli\u003eSingh MK, Dominy BN (2012) The Evolution of Cefotaximase Activity in the TEM \u0026beta;-Lactamase. J Mol Biol 415:205-220. https://doi.org/10.1016/j.jmb.2011.10.041\u003c/li\u003e\n\u003cli\u003eKumar D, Singh AK, Ali MR, Chander Y (2014) Antimicrobial Susceptibility Profile of Extended Spectrum \u0026beta;-Lactamase (ESBL) Producing Escherichia coli from Various Clinical Samples. Infect Dis (Auckl) 7:IDRT.S13820. https://doi.org/10.4137/IDRT.S13820\u003c/li\u003e\n\u003cli\u003eDrawz SM, Bonomo RA (2010) Three Decades of \u0026beta;-Lactamase Inhibitors. Clin Microbiol Rev 23:160-201. https://doi.org/10.1128/CMR.00037-09\u003c/li\u003e\n\u003cli\u003eSalverda MLM, De Visser JAGM, Barlow M (2010) Natural evolution of TEM-1 \u0026beta;-lactamase: experimental reconstruction and clinical relevance. FEMS Microbiol Rev 34:1015-1036. https://doi.org/10.1111/j.1574-6976.2010.00222.x\u003c/li\u003e\n\u003cli\u003ePimenta AC, Fernandes R, Moreira IS (2014) Evolution of drug resistance: insight on TEM \u0026beta;-lactamases structure and activity and \u0026beta;-lactam antibiotics. Mini Rev Med Chem 14:111-122. https://doi.org/10.2174/1389557514666140123145809\u003c/li\u003e\n\u003cli\u003eTooke CL, Hinchliffe P, Bragginton EC, Colenso CK, Hirvonen VHA, Takebayashi Y, Spencer J (2019) \u0026beta;-Lactamases and \u0026beta;-Lactamase Inhibitors in the 21st Century. J Mol Biol 431:3472-3500. https://doi.org/10.1016/j.jmb.2019.04.002\u003c/li\u003e\n\u003cli\u003eCant\u0026oacute;n R, Novais A, Valverde A, Machado E, Peixe L, Baquero F, Coque TM (2008) Prevalence and spread of extended-spectrum \u0026beta;-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect 14:144-153. https://doi.org/10.1111/j.1469-0691.2007.01850.x\u003c/li\u003e\n\u003cli\u003eM\u0026ouml;ller M, Denicola A (2002) Protein tryptophan accessibility studied by fluorescence quenching. Biochem Mol Biol Educ 30:175-178. https://doi.org/10.1002/bmb.2002.494030030035\u003c/li\u003e\n\u003cli\u003eKumar V, Lakshman PK, Prasad TK, Manjunath K, Bairy S, Vasu AS, Ganavi B, Jasti S, Kamariah N (2024) Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening. Heliyon 10(1):e23864. https://doi.org/10.1016/j.heliyon.2023.e23864\u003c/li\u003e\n\u003cli\u003eMukherjee SK, Mandal RS, Das S, Mukherjee M (2018) Effect of non-\u0026beta;-lactams on stable variants of inhibitor-resistant TEM \u0026beta;-lactamase in uropathogenic Escherichia coli: implication for alternative therapy. J Appl Microbiol 124:667-681. https://doi.org/10.1111/jam.13671\u003c/li\u003e\n\u003cli\u003eSwain BC, Mukherjee SK, Rout J, Sakshi, Mishra PP, Mukherjee M, Tripathy U (2020) A spectroscopic and computational intervention of interaction of lysozyme with 6-mercaptopurine. Anal Bioanal Chem 412:2565-2577. https://doi.org/10.1007/s00216-020-02483-1\u003c/li\u003e\n\u003cli\u003eMandal P, Bardhan M, Ganguly T (2012) Spectroscopic investigations to reveal the nature of interactions between the haem protein myoglobin and the dye rhodamine 6G. Luminescence 27:285-291. https://doi.org/10.1002/bio.1348\u003c/li\u003e\n\u003cli\u003eMishra PP, Patel S, Datta A (2006) Effect of Increased Hydrophobicity on the Binding of Two Model Amphiphilic Chlorin Drugs for Photodynamic Therapy with Blood Plasma and Its Components. J Phys Chem B 110:21238-21244. https://doi.org/10.1021/jp0615858\u003c/li\u003e\n\u003cli\u003eBertucci C, Pistolozzi M, De Simone A (2010) Circular dichroism in drug discovery and development: an abridged review. Anal Bioanal Chem 398:155-166. https://doi.org/10.1007/s00216-010-3959-2\u003c/li\u003e\n\u003cli\u003eMiani A, Raugei S, Carloni P, Helfand MS (2007) Structure and Raman Spectrum of Clavulanic Acid in Aqueous Solution. J Phys Chem B 111:2621-2630. https://doi.org/10.1021/jp066135u\u003c/li\u003e\n\u003cli\u003eChakraborty B, Mitra P, Basu S (2015) Spectroscopic exploration of drug-protein interaction: a study highlighting the dependence of the magnetic field effect on inter-radical separation distance formed during photoinduced electron transfer. RSC Adv 5:81533-81545. https://doi.org/10.1039/C5RA13575C\u003c/li\u003e\n\u003cli\u003eMukherjee SK, Mukherjee M, Mishra PP (2021) Impact of Mutation on the Structural Stability and the Conformational Landscape of Inhibitor-Resistant TEM \u0026beta;-Lactamase: A High-Performance Molecular Dynamics Simulation Study. J Phys Chem B 125:11188-11196. https://doi.org/10.1021/acs.jpcb.1c05988\u003c/li\u003e\n\u003cli\u003eAbdul Samad F, Suliman BA, Basha SH, Manivasagam T, Essa MM (2016) A Comprehensive In Silico Analysis on the Structural and Functional Impact of SNPs in the Congenital Heart Defects Associated with NKX2-5 Gene - A Molecular Dynamic Simulation Approach. PLoS ONE 11:e0153999. https://doi.org/10.1371/journal.pone.0153999\u003c/li\u003e\n\u003cli\u003eM\u0026aacute;tyus L, Sz\u0026ouml;llősi J, Jenei A (2006) Steady-state fluorescence quenching applications for studying protein structure and dynamics. J Photochem Photobiol B 83:223-236. https://doi.org/10.1016/j.jphotobiol.2005.12.017\u003c/li\u003e\n\u003cli\u003eTopală T, Bodoki A, Oprean L, Oprean R (2014) Bovine serum albumin interactions with metal complexes. Med Pharm Rep 87:215-219. https://doi.org/10.15386/cjmed-357\u003c/li\u003e\n\u003cli\u003eGhisaidoobe A, Chung S (2014) Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on F\u0026ouml;rster Resonance Energy Transfer Techniques. Int J Mol Sci 15:22518-22538. https://doi.org/10.3390/ijms151222518\u003c/li\u003e\n\u003cli\u003eChib R, Butler S, Raut S, Shah S, Borejdo J, Gryczynski Z, Gryczynski I (2015) Effect of quencher, denaturants, temperature and pH on the fluorescent properties of BSA protected gold nanoclusters. J Luminescence 168:62-68. https://doi.org/10.1016/j.jlumin.2015.07.030\u003c/li\u003e\n\u003cli\u003eBardhan M, Chowdhury J, Ganguly T (2011) Investigations on the interactions of aurintricarboxylic acid with bovine serum albumin: Steady state/time resolved spectroscopic and docking studies. J Photochem Photobiol B 102:11-19. https://doi.org/10.1016/j.jphotobiol.2010.08.011\u003c/li\u003e\n\u003cli\u003eRoss PD, Subramanian S (1981) Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 20:3096-3102. https://doi.org/10.1021/bi00514a017\u003c/li\u003e\n\u003cli\u003eZhao Q, Tao J, Uppal JS, Peng H, Wang H, Le XC (2019) Nucleic acid aptamers improving fluorescence anisotropy and fluorescence polarization assays for small molecules. TrAC Trends Anal Chem 110:401-409. https://doi.org/10.1016/j.trac.2018.11.018\u003c/li\u003e\n\u003cli\u003eLee MM, Peterson BR (2016) Quantification of small molecule-protein interactions using FRET between tryptophan and the pacific blue fluorophore. ACS Omega 1:1266-1276. https://doi.org/10.1021/acsomega.6b00356\u003c/li\u003e\n\u003cli\u003ePezzotti G (2021) Raman spectroscopy in cell biology and microbiology. J Raman Spectrosc 52:2348-2443. https://doi.org/10.1002/jrs.6204\u003c/li\u003e\n\u003cli\u003eGill M, McCully ME (2019) Molecular dynamics simulations suggest stabilizing mutations in a de novo designed \u0026alpha;/\u0026beta; protein. Protein Eng Des Sel 32:317-329. https://doi.org/10.1093/protein/gzaa005\u003c/li\u003e\n\u003cli\u003eFantini S, Lisi S, De Los Rios P, Cattaneo A, Pastore A (2020) Protein Structural Information and Evolutionary Landscape by In Vitro Evolution. Mol Biol Evol 37:1179-1192. https://doi.org/10.1093/molbev/msz256\u003c/li\u003e\n\u003cli\u003eGaldadas I, Qu S, Oliveira ASF, Olehnovics E, Mack AR, Mojica MF, Agarwal PK, Tooke CL, Gervasio FL, Spencer J, Bonomo RA, Mulholland AJ, Haider S (2021) Allosteric communication in class A \u0026beta;-lactamases occurs via cooperative coupling of loop dynamics. ELife 10:e66567. https://doi.org/10.7554/eLife.66567\u003c/li\u003e\n\u003cli\u003eYang J, Naik N, Patel JS, Wylie CS, Gu W, Huang J, Ytreberg FM, Naik MT, Weinreich DM, Rubenstein BM (2020) Predicting the viability of beta-lactamase: How folding and binding free energies correlate with beta-lactamase fitness. PLoS ONE 15:e0233509. https://doi.org/10.1371/journal.pone.0233509\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 To 3 are available in the Supplementary Files section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eVariation in a-helix% of TEM-1(wild type protein) and mutant \u0026beta;‑lactamases (M184, M203 and M210) with increase concentration of Ceftazidime (CAZ).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e[\u0026beta;‑lactamases]M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e[CAZ]M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u0026alpha; \u0026ndash; helix (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTEM-1(~5x10\u003csup\u003e-6\u003c/sup\u003e M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e59.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~0.5 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e41.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e27.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.5 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e26.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~2.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e20.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eM184(~5x10\u003csup\u003e-6\u003c/sup\u003e M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e44.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~0.5x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e42.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e34.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.5 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e33.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~2.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e14.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eM203(~5x10\u003csup\u003e-6\u003c/sup\u003e M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e69.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~0.5 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e55.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e49.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.5 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e30.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~2.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e10.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eM210(~5x10\u003csup\u003e-6\u003c/sup\u003e M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e79.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~0.5 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e76.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e53.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~1.5 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e40.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 212px;\"\u003e\n \u003cp\u003e~2.0 x10\u003csup\u003e-5\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e33.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TEM β-lactamase mutants, absorbance spectroscopy, fluorescence quenching, fluorescence anisotropy, Van't Hoff plot, Molecular dynamics simulation","lastPublishedDoi":"10.21203/rs.3.rs-7504842/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7504842/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003ePurpose\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study investigated the bioactive interactions of ceftazidime (CAZ) to TEM β-lactamase variants with distant site mutations isolated from clinical settings to explore the cause of their selection and dissemination due to empirical use of β-lactams.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBinding interactions of CAZ with wild-type and mutant (M184, M203, M210) TEM β-lactamases were recorded by UV\u0026ndash;Vis absorption and fluorescence spectra (280\u0026ndash;600 nm) at 298\u0026ndash;311 K with inner filter correction. Binding constants were determined using Stern\u0026ndash;Volmer and modified Stern\u0026ndash;Volmer equations. Fluorescence lifetime, quantum yield and anisotropy measurements ascertained energy transfer and microenvironmental changes. Structural alterations and interaction energetics were evaluated using circular dichroism, Raman spectroscopy, F\u0026ouml;rster resonance energy transfer and Molecular dynamics simulation (100 ns) study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBiophysical experimentation indicated facilitated binding of CAZ to the active site of the mutants than the wild type. The CAZ-TEM β-lactamase mutant interactions were predominantly hydrophobic compared to H-bonding and van der Waals forces in the CAZ-wild type complex. Additionally, structural alteration to justify more rigid binding of CAZ to the mutants in contrast to the wild type enzyme was established \u003cem\u003ein silico\u003c/em\u003e. Acquisition of distant site mutations with respect to the active site of TEM β-lactamases rendered conformational flexibility to accommodate CAZ was evidenced.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTherefore, this study established that selection of far site mutations in TEM β-lactamases can indirectly contribute to β-lactam resistance by optimizing substrate binding dynamics in response to rampant usage of the antibiotics and provided a new dimension towards future drug development.\u003c/p\u003e","manuscriptTitle":"Distant site mutations in clinical TEM β-lactamase variants enhance non-covalent binding to ceftazidime: Insights from biophysical and in silico investigations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 17:39:05","doi":"10.21203/rs.3.rs-7504842/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e09f33f-56a3-4826-8e5c-ec5110b0b058","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-19T14:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-01 17:39:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7504842","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7504842","identity":"rs-7504842","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}