Background: The angiotensin-1-converting enzyme (ACE) is a zinc protease unique in its chloride ion dependence.Results: Key substrate interactions with the catalytic site affect the structure and thermodynamics of ACE, which modulate chloride ion-dependent hydrolysis.Conclusion: Substrate composition influences chloride affinity in ACE.Significance: The interdependence of ACE substrate composition, structure, and thermodynamics influence its chloride ion activation in biology. 522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg Angiotensin I-converting enzyme ACE–3. The sXXH zinc-binding motif residing on the α15 helix , and a water molecule , whereas for captopril, the difference is larger at low chloride concentration (20 mm)  . The var403 and Lys118 was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work provides a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.In this study, key single point mutations were generated to investigate the mechanism of chloride activation and substrate specificity. Activity of these mutants was characterized using an isothermal titration calorimetry (ITC)-based assay providing in depth information on the enzyme thermodynamic and kinetic parameters under varying chloride concentrations. Furthermore, the high resolution x-ray crystal structures of these mutants were determined, revealing the key molecular interactions involved in chloride binding. Together, these data identified the close relationship between the nature of the substrate and chloride activation through the chloride 2 pocket and the S2 subsite. The role for the chloride 1 pocket remains elusive and could be mainly structural. A key salt bridge specific to C-domain ACE between Glul-Phe-l-His-Leu (Z-FHL) was purchased from Bachem AG .All chemicals were purchased from Merck and Sigma-Aldrich. Benzyloxycarbonyl-N-glycosylated version of human tACE, tACEΔ36-g13sol  and purified to homogeneity as described previously , 19.m HEPES, pH 7.5, 0.1 mm PMSF) mixed with an equal volume of reservoir solution  and suspended above the well as a hanging drop. Diffraction quality crystals of C-domain mutants appeared after ∼3–5 days. The R522K·captopril complex was obtained by co-crystallization with a 1 mm concentration of the ligand.Crystals were obtained with 1 μl of the C-domain mutant sample . 25% PEG 3350 was added to the drop as a cryoprotectant to keep the crystal at constant temperature (100 K) under the liquid nitrogen jet during data collection. For each mutant, 100–150 images were collected by using a PILATUS-2M, -6M  or a Quantum-315 CCD  detector. Raw data images were processed and scaled with XDS , MOSFLM 1O8A . Five pelidation . Water mLPROBITY . CrystalZ-FHL solutions containing varying amounts of NaCl (0–1 m) prepared using Milli-Q distilled H2O (chloride concentration <0.06 mm). All substrates used were HPLC-purified (>98% purity), so any chloride or chloride salts would elute with the breakthrough peak; hence, the effects on chloride concentrations are negligible. Assays were performed using a protocol adapted from that described previously (m TAPSO (pH 7.5)). Milliunits of ACE activity, with 1 unit defined as 1 nmol of HL produced/min/ml (min−1·ml−1) at 37 °C in assay buffer, were calculated, divided by the total number of μg in the reaction volume, and expressed as specific activity (milliunits·μg−1). Substrate concentrations used were ≥5 times mK, and the total percentage hydrolysis in each reaction was kept below 20% in order to approximate first order rates (kcat/mK). The measure of chloride binding (dK(app)) was calculated from activation plots of specific activity versus the logarithm of chloride concentration, with the resultant curves analyzed via non-linear regression using a sigmoidal dose-response curve with the formula,min is the specific activity in the absence of NaCl, SAmax is the maximum specific activity upon titration with chloride, X is the logarithm of chloride concentration, Y is the response (specific activity), and EC50 is the X value when the response is halfway between SAmax and SAmin.Chloride titrations were performed using HHL and eviously , with thm) were dialyzed extensively against 3× 1 liter of reaction buffer, which consisted of 50 mm TAPSO buffer (pH 7.5), 10 μm ZnSO4, and either 0, 20, or 300 mm NaCl.Enzyme samples of high concentration , or by equilibrating against a 1-ml G10 Sephadex desalting column (for angiotensin I). Angiotensin I  was dissolved in dialysate at high concentration then passed through a 1-ml G10 column  under gravity. 200-μl fractions were collected, and absorbance was measured at 275 nm. These were pooled, and the final concentration was determined spectrophotometrically via absorbance at 275 nm, using the empirically calculated extinction coefficient of 1280 200 microcalorimeter and according to the guidelines in the iTC200 microcalorimeter user manual provided by MicroCal. Buffer conditions for both enzyme and lisinopril consisted of 50 mm TAPSO buffer (pH 7.5), 10 μm ZnSO4, and either 0, 20, or 300 mm NaCl, prepared using Milli-Q distilled H2O (chloride concentration <0.06 mm). The reaction cell was maintained at 20 °C, with the lisinopril concentration in the syringe (800–1400 μm) at 10 times the enzyme concentration in the cell (8–12 μm) to account for lisinopril dilution (∼6-fold) and ensure a complete 1:1 binding curve. Binding assays were performed with every preparation of enzyme used in the kinetic assays; this served as an active site titration to determine active enzyme concentration in order to improve the accuracy of the kcat  values obtained in those experiments. The dK(app), ΔG, ΔH, and ΔS values for lisinopril binding were calculated using Origin 7 with the iTC200 MicroCal Software Addon.Assays were performed using an iTC200 microcalorimeter , with raw data either extracted for custom calculations using Microsoft Excel or analyzed using Origin 7 software with a proprietary MicroCal analysis module. Assays were performed at 37 °C using an adapted version of the progress curve method described by Stockbridge and Wolfenden  and each time point (t) and then dividing by the total integrated area gives a percentage of the product formed at time t. Multiplying this fraction at each time point by the total substrate concentration at t = 0 (S0) relates it to molar concentration. The formula describing this relationship is as follows,P(t) (product formed) versus t (time).All ITC experiments were performed using an iTColfenden , where teviously . Given tW is the Lambert W(x) function  are reported as the mean of the three separate determinations along with the S.E. value.Progress curves were generated and analyzed according to functions described by Golicnik . The temfunction . Golicnifunction  evaluateG, ΔH, and ΔS) for the hydrolysis of substrates  were determined using the kinetic data (kcat/mK) and the raw data from each of those assays. The Gibbs free energy (ΔG) was calculated by the formula ΔG = −RT ln(kcat/mK), with R being the gas constant  and T being assay temperature (310 K). The apparent enthalpy (ΔHapp) was determined for each assay via integration of the area under the curve using the following formula.Hint) was calculated using the relationship, ΔHapp = ΔHint + nH × ΔHion . Incorporation of ΔG and ΔHint into ΔG = ΔHint − TΔS allows calculation of −TΔS, which is the entropy as a function of temperature. These values were calculated separately for each assay, which was done in triplicate as a minimum, with values shown as mean and S.E.The thermodynamic parameters (ΔB). The consequences appeared to be localized around the mutated sites, which were designed to investigate the role of these specific residues in the chloride-dependent enzymatic activity. Mutations R522K (ligand-free) and R522Q showed the loss of the chloride ion in proximity to the active site  but is expected to be very mobile, as illustrated by the lack of electron density . The malonic acid is coordinating with the same Arg221 on one side and mimicking the interactions previously made by Asp465 on the other side, including the salt bridge with Arg468 and hydrogen bonds with Tyr520 and Ile521. Noticeably, loop Ser434–Ser439 was visible for the first time in a C-domain crystal structure (B) with clear electron density observed for this flexible loop region.A high resolution data set was collected for the D465T mutant. The native Asptructure B with cl522 in the native C-domain, along with Tyr224 and a water-mediated interaction to Trp220 (C). The R522K mutant was crystallized with and without captopril . Whereas the Cl− ion is not visible in the ligand-free structure, the ion was clearly visible in the bound complex. There is no evidence from the electron density map of the R522K·captopril structure that the chloride is linked to Lys522 (C) with the amino group located more than 4 Å away from a water in the ion's hydration shell. The chloride ion is coordinated by the hydroxyl group of Tyr224 and the surrounding water molecules linked to Trp220, Trp469, and Asp465. Importantly, Lys522 now forms a salt bridge with Glu411, which is part of the zinc coordination motif and thus involved in the enzyme catalytic activity. Lys522 is also within the distance of a cation-π interaction with Tyr523, thus further directing the side chain away from the Cl− ion. It should be added that captopril is present at the active site in the same position as that of the wild-type C-domain·captopril complex structure . The position of the residues within the chloride pocket remains unchanged, and Lys522 is therefore making interactions similar to those described for the ligand-bound structure. An additional water-mediated contact was seen with Glu411, reinforcing the link with the catalytic pocket.The chloride ion is directly coordinated by Argo Trp220 C. The R5o Lys522 C with thtructure . Althougf Tyr224 D. The poE), with Gln522 making strong hydrogen bonds with the backbone of residues Pro519, Tyr520, and Ile521 . Overall, the enthalpy (ΔH) is positive and very similar between the N- and C-domains as well as between substrates for both domains, indicating that the enthalpic contributions are very similar between the domains. What is also evident for all substrates with both domains is the larger contribution to ΔG by entropy (−TΔS) than enthalpy (ΔH), showing that binding is most likely driven by hydrophobic interactions, with desolvation of substrate and structural changes being possible contributions.In evaluating the effect of chloride on the thermodynamic profile of the C- and N-domains, the thermodynamic parameters were determined in the presence and absence of chloride , A and BA and B). For all three substrates, there were negligible changes in the ΔH values, suggesting that increased activity in the presence of chloride is entropically driven. The fairly consistent ΔH, with and without chloride, between substrates suggests that the main interactions that form part of the catalytic mechanism are preserved, which is consistent with the observation that catalysis does occur in the absence of chloride.There was a significant decrease in ΔG, associated with reduced catalysis, in the absence of chloride for C- and N-domains using AngI, HHL, and Z-FHL , A and BG, ΔH, and −TΔS  between 0 mm NaCl and the concentration of maximal activity  were calculated for both domains (C). For the C-domain, the greatest change in ΔG is observed with HHL, with reduced change seen for Z-FHL and even less for AngI. These changes in free energy are almost completely entropically-driven, as evidenced by the −TΔS values showing an almost identical trend. There is also almost no change in the enthalpy for AngI, with minor increases for HHL  and Z-FHL approaching 0.5 kcal/mol . The cleavage of a peptide bond would presumably lead to a lowering of enthalpy by 2–5 kcal/mol. However, it can be assumed that the conformational changes suggested by the negative −TΔΔS values would result in the formation of sufficient interactions, presumably stabilizing in nature, to counterbalance the effect of peptide bond cleavage and result in an overall slightly positive ΔΔH. The entropy-linked increase in enthalpy is probably the result of hydrophobic forces, either via desolvation of hydrophobic groups upon binding of substrate or via the formation of new bonds with a structural change. By contrast, the corresponding ΔΔG, ΔΔH, and −TΔΔS values for the N-domain present far less variation between the different substrates. The ΔΔG and −TΔΔS for HHL are considerably less negative (∼2-fold), whereas, in comparison, those for Z-FHL are relatively unchanged, with the AngI values being slightly more negative than those for the C-domain. The larger entropic shift for AngI hydrolysis with the N-domain is driven by a ∼0.5 kcal/mol increase in the enthalpy, which could be a loss of specific interactions due to variability of interactions with AngI between the two domains.In order to better evaluate the chloride-dependant shifts in thermodynamic data, the changes in Δ domains C. For thdK(app) values were compared with the -fold increase in kcat/mK between 0 mm NaCl and maximal activity for HHL, Z-FHL, and AngI for both domains (D). The dK(app) and -fold increase in activity for the C- and N-domains are considerably higher for HHL. This indicates that the chloride dependence mechanisms of the C- and N-domains are most pronounced with the shorter HHL substrate and modulated by the increased length of the Z-FHL and AngI substrates.The largest variation in energetic contributions between domains is seen with HHL, suggesting that the difference in substrate composition, and hence interactions, compared with Z-FHL and AngI could be responsible for the observed shift. To highlight this variation, the  domains D. The Kdm NaCl is shown in A and B. At 0 mm NaCl, all substrates show an approximate halving of activity relative to the C-domain (A), which can be attributed to increased mK values , which compares favorably with results previously reported for a R186Q mutant (kcat/mK for AngI are due to the mK for R186H (0.095 mm) being almost 3-fold higher than for the C-domain (0.036 mm), with very little change in kcat . This is in contrast to Z-FHL, where the change is due to an almost 2-fold increase in kcat from the C-domain (282.0 s−1) to R186H (509.5 s−1), with a concomitant minor increase in mK . HHL hydrolysis showed a similar pattern to AngI, with an increased mK from the C-domain to R186H, yet a change in kcat with Z-FHL is observed, suggesting that interactions in the chloride 1 pocket play a role in mediating both substrate binding and catalysis.The chloride 1 pocket was proposed to be involved in C-terminal stabilization of substrates , 33, andC-domain A, which m values . In the is 40.4% B, which Q mutant . These vdK for R186H (132 ± 6.2 nm) being ∼3-fold higher than for the C-domain (45.5 ± 4.24 nm) at 0 mm NaCl, as well as ∼3-fold lower at 20 mm NaCl  (H) for the substrates yet not more than 11% change in the overall ΔH values (C). This suggests that there is no significant variation in interactions and that the effect is probably due to minor structural changes. The lack of a distinct effect on activity-associated chloride binding in the chloride 1 pocket of the R186H mutant supports the theory that the chloride 2 pocket is the main mechanistic effector of chloride dependence and that the chloride 1 pocket exerts its influence primarily via affinity modulation in the prime binding site. This also serves as a key differentiator in chloride-mediated substrate/inhibitor affinity between the domains.Supporting this, a large change in binding affinity with lisinopril for R186H relative to the C-domain is also observed, with the ctively) . This fuH values C. This s182 as (N-domain) His164 does with Trp160 and with Arg489 showing similar dual coordination of the Trp279 carbonyl. However, this would not explain why R186Q . Tyr520, Ile521, Arg522, and Tyr523 are the first four residues on the C-terminal end of the α23 helix (Tyr520–Ala541), with Pro519 representing the end of the helix and the beginning of a highly variable loop. However, this region of the α23 helix is within subdomain II of both domains, a region predicted to have little major structural variation relative to the proposed hinge region that is part of subdomain I  does not interact with Arg446 (Arg468 in the C-domain), but Arg446 does hydrogen-bond with Thr496, which corresponds to Val518 in the C-domain. Both Val518, in the S1 subpocket, and His513, which interacts with Tyr523  and the activity at 0 mm NaCl as a percentage of maximal activity (%SAmax) for D465T with both HHL and Z-FHL were lower than those of the C-domain. An increase in dK(app) for Z-FHL and a decrease for HHL with D465T was also observed. The location of Asp465 on the side of the chloride 2 pocket opposite from the proposed chloride channel area precludes any direct interaction with the chloride ion and involvement in the chloride channel mechanism. These results may be explained by the fact that Asp465 stabilizes the C-terminal end of helix α23 and the variable loop via hydrogen bonding with the primary amides of Tyr520 and Ile521. The Thr in this position is capable of fewer potential hydrogen bonds . Its effect on %SAmax and dK(app) is probably due to structural destabilization, illustrated by the rearrangement of Trp469 , which suggests that structural rotation or movement around the C-terminal end of helix α23, which contains Arg522 and Tyr523, moderates not only activity but chloride affinity.The chloride titration data determined for HHL and Z-FHL using the D465T mutant  support 522 and Tyr523 is likely to influence chloride activation. The crystal structure of native C-domain showed the possible cation-π bond between these two residues, whereas the structures of the two 522 mutants demonstrated little structural change apart from the loss of chloride coordination.Thus, the interaction between ArgdK(app) values for HHL and Z-FHL (A), the largest increases relative to the C-domain are seen for HHL with R522K (7-fold) and R522Q (9-fold). A similar trend is seen with the dK(app) values for Z-FHL and AngI (despite the lack of R552Q dK(app)) although not to the same degree as for HHL.Given these potential interactions, the kinetic data obtained for R522K and R522Q mutants relative to C-domain can be assessed. Comparing the nd Z-FHL A, the la522 with a lysine or a glutamine causes a decrease in chloride affinity, in agreement with the structural data. Furthermore, R522K and R522Q follow the same dK(app) trend as the C-domain (AngI < Z-FHL < HHL) although proportionally higher than the C-domain for each. This indicates that substrate interactions are somehow moderating access and binding of the chloride ion. This is confirmed by the presence of chloride in the pocket of R522K only when a ligand is bound at the active site (and captopril is a peptide mimetic). Substrate binding would cause local restructuring to interact with the catalytic site and thereby also close access to the chloride pocket. The sulfhydral group of captopril coordinates the zinc ion, thereby causing a loss of the hydrogen bond from Tyr523 seen in the tACE·lisinopril complex . At 0 mm NaCl, R522Q shows a marked increase in activity over both C-domain and R522K with Z-FHL and AngI (B), suggesting that additional interactions for the 522 position beyond chloride coordination are important. At 20 mm NaCl, there is an increase in activity for both R522K and R522Q with larger substrates that interact more tightly with the S1 and S2 active site pockets (C). Although R522K shows only a marginal increase over HHL (AngI (15.6%) > Z-FHL (9.3%) > HHL (0.1%)) relative to C-domain at 20 mm NaCl, there is a marked increase for R522Q (AngI (87.7%) > Z-FHL (27.5%) > HHL (0.36%)) to the point where R522Q activity is almost as high as C-domain activity with AngI. This would indicate that the bonding pattern is different between R522K and R522Q and that interactions of longer substrates have an effect on either the chloride 2 pocket or a key catalytic mechanism. To further probe this trend, the contributions of mK and kcat to the catalytic efficiency of R522Q and R522K are evaluated. For HHL, the mechanistic lack of activation is demonstrated by the low variation in mK but marked differences in the kcat, where values are similar at 0 mm chloride and only C-domain showing an increase at 20 mm. Interestingly, R522Q shows a 5-fold lower mK for Z-FHL at 0 mm NaCl than C-domain, yet this trend is reversed at 20 mm, with the mK for R522Q being ∼2-fold higher than that for the C-domain. The data show that mK for R522K and R522Q with Z-FHL is higher at 20 mm than at 0 mm, a reversal of the general trend for the C-domain with all substrates. Both mutants show less than 2-fold increases in kcat from 0 to 20 mm NaCl, versus the greater than 3-fold increase observed for the C-domain. Thus, the large decrease in activation for R522Q and R522K with Z-FHL is influenced by the increased mK as well as the reduced kcat associated with the presence of chloride, in contrast to HHL, which is predominantly influenced by kcat. Interestingly, a similar lack of variance is seen in mK values for R522Q and R522K with AngI and greater variation in kcat. The increases in mK observed for all substrates with R522K and, to a lesser degree, R522Q indicate that coordination by chloride in this pocket affects substrate affinity as well as catalysis, suggesting some structural movement around helix α23. The most noticeable effect of the R522Q mutation is the greater kcat values that increase with substrate length and are even higher than C-domain for AngI. Taking into account the structural data, it is likely that the lack of chloride coordination allows for more freedom of movement at the S1 recognition site, particularly for Tyr523, thus resulting in more efficient catalysis. It is therefore unlikely that the cation-π interaction is favored over the hydrogen bonds between Lys522 and Glu411 and between Gln522 and α23, respectively.Other interactions in the chloride 2 pocket are the salt bridge between Lysan R522Q . To bettC-domain , B and Cand AngI B, sugges pockets C. AlthouD). For R522Q, a reduced endothermic change in enthalpy is seen for HHL  and ZHFL  relative to the C-domain , which would indicate the loss of an interaction(s) with the binding site. That the ΔΔH values do not exceed the minimum hydrogen bonding energy  might be due to differing hydration effects associated with the different residue counteracting the enthalpic change.The changes in thermodynamic profiles in the presence of chloride for R522Q and R522K were evaluated D. For R5G and the kcat/mK is lower than C-domain and R522Q at both 0 and 20 mm NaCl. Given the assertions that Lys neither coordinates chloride nor cation-π-bonds with Tyr523, the interactions of Z-FHL in the S1 and S2 pockets may be compensating via minor structural shifts. The endothermic shift in enthalpy observed with AngI for R522K is accompanied by a relatively large shift in free energy  similar in magnitude to that in the C-domain , which actually results in a larger −TΔΔS value due to the more endothermic enthalpy shift. There is a 5-fold increase in kcat/mK from 0 to 20 mm NaCl, resulting in the observed ΔΔG, although these values are considerably lower than in the C-domain. The modeling of AngI bound to C-domain  is within the distance (5.5 Å) of the Arg522 hydration shell. These important interactions may account for the chloride-mediated ΔΔG as R522K and the catalytic site overall (through bonding with Glu411 and α17) lose the stability provided by the chloride ion.For R522K, there is a different pattern with the enthalpy changes, with HHL  showing an exothermic shift relative to the C-domain 0.36 kcal/mol), AngI  showing an endothermic shift relative to the C-domain , and Z-FHL relatively unchanged. As with R522Q, an exothermic shift would suggest a bond breaking for HHL, which would be expected with the lack of direct chloride coordination. The lack of variation in enthalpy with Z-FHL for R522K is interesting, considering that there is no ΔΔC-domain  based on kcal/mol522 toward the catalytic channel, thereby promoting catalysis of substrate. Substrate interactions within the S1/S2 pockets affect positioning of this complex and provide a rationale whereby substrate binding modulates chloride affinity and hence its own catalysis.Chloride coordination appears necessary to stabilize Arg403 may act as an ionic gate mediating access of chloride to the chloride 2 pocket (381 in the N-domain (dK(app) for chloride binding with both HHL and Z-FHL relative to the C-domain  values showed 29- and 15-fold increases for HHL (4.7 mm) and Z-FHL (11.4 m) over the N-domain . Furthermore, substrate interactions in the S2 subpocket where Glu403 resides might disrupt any ion gating, as evidenced by the large discrepancy in dK(app) seen between HHL and Z-FHL for the C-domain, which was largely abolished with E403R.The E403R mutation was generated to investigate a proposed chloride channel, where Glu2 pocket . For theN-domain . The E40C-domain . This efdK(app) varies with different substrates, suggesting that chloride coordination is affected, but changes in overall activity and degrees of activation are also seen, thus highlighting further functions for Glu403. The E403R kcat/mK values are shown relative to the C-domain at 0 mm NaCl (A) and at maximal activity (B) for HHL, Z-FHL, and AngI. The kcat/mK values for E403R with all substrates at maximal activity are generally lower relative to the C-domain, whereas at 0 mm NaCl, they are all higher. This indicates that Glu403 affects activity as well as potentially mediating access to the chloride 2 pocket. Glu403 makes a salt bridge with Lys118 in the native structure  of the peptide . Lys118 proved less stable . Thus, E403R and the N-domain lack this stabilizing salt bridge and show lower dK(app) values for chloride binding . The ΔΔG values are reduced for all substrates relative to the C-domain, which is due to the observed increase in kcat/mK at 0 mm NaCl and decrease at 20 mm NaCl (A and B). That the pattern and magnitude of the enthalpies (ΔΔH) for the substrates correlates well with the C-domain indicates that there should be no major structural rearrangement. The lack of significant enthalpy changes do not discount minor structural differences, which are evidenced by less favorable changes in entropy (−TΔΔS). These minor structural effects would help explain the mK and kcat data for E403R relative to C-domain  relative to C-domain (282.00 and 7.38 s−1), which is countered by higher mK values in the presence of chloride for E403R (0.530 and 0.061 mm) over the C-domain (0.120 and 0.036 mm). These result in lower maximal kcat/mK values with both substrates for E403R but serve to confirm that some structural variation is present. The HHL substrate does not appear to interact strongly within the S2 region, and it is likely that its affinity is predominantly affected by S1′/S2′ interactions.Further evidence of the stabilizing role of the GluC-domain C. The ΔΔ mm NaCl , A and BC-domain . E403R s403 in the C-domain via its interaction with Lys118 within the hinge-bending region rather than a functional ion-gated channel. The salt bridge serves to reduce the affinity of chloride within the chloride 2 pocket of C-domain as well as affect the structural architecture of the S2 pocket. Substrate interactions within this pocket, exemplified by Z-FHL and to a greater extent AngI, appear to disrupt the Glu403-Lys118 interaction and allow for improved chloride binding. The absence of interaction in the N-domain means that the chloride 2 pocket would have a higher affinity for chloride, even in the absence of any substrate interaction within the S2 pocket. The size of the substrate and, more importantly, how far it extends into the non-prime subsites has been shown to be important where substrate interactions in the S2 pocket modulate chloride affinity directly in the C-domain of ACE. These interactions are likely to be propagated via structural perturbations around the active site rather than any specific interaction, which is consistent with the high degree of variability in ACE substrate identity.These results provide evidence for a stabilizing role for Glu522 interacts indirectly with Glu411 and Tyr523, thus affecting the catalytic site by stabilizing the enzyme-substrate transition state intermediate. Importantly, the affinity of chloride coordinating Arg522 is moderated by structural constraint via the Glu403-Lys118 salt bridge in the C-domain of ACE, which is a point of major difference with the N-domain. Arg522 is part of α23, which includes other important residues for recognition of the substrate C-terminal carboxylate along with Lys511 situated on the 310 H6 helix . The P1′ and P2′ residue interactions within their respective pockets have an unfavorable influence on chloride affinity, presumably via interaction or non-interaction with Tyr523 and Tyr520 (dK(app) for chloride binding; thus, they are likely to moderate chloride affinity via interaction with the S2 pocket. This may be mediated by disruption of the Glu403-Lys118 salt bridge. It is possible that the greater length of AngI has a significant structural effect on the chloride pocket, which may also account for the relatively lower kcat values.The effect of substrate composition on activity can be inferred from the kinetic and structural data. The tripeptide HHL shows a highly chloride-dependent profile exemplified by a high d Tyr520 . Longer et al.  and fibrosis (N-selective ACEi).Key amino acid mutations in the C-domain of ACE showed marked effects on chloride dependence when evaluated using kinetic and thermodynamic data determined for the hydrolysis of HHL, Z-FHL, and AngI under various chloride concentrations. The interpretation was complemented with the molecular details provided by the determination of the crystal structures for four of the mutants. A model of interactions in the chloride 1 pocket was developed to explain how chloride binding might modulate Lys


Due to in (APP) . HoweverUsing X-ray crystallography we solved the structure of the APP-E1-domain  and togeHere we present the structural and biochemical characterization of the APP-E1-domain showing that the growth-factor-like domain (GFLD) and the copper-binding domain (CuBD) form one closed structural and hence functional entity. We could show concentration dependent self-dimerization of the E1 domain at high and the heparin dependent dimerization at low protein concentrations and set up a model of dimerization, which was also based on crystallographic data. We additionally could prove this model using our E1 mutants.We could nicely show the dimerization of the APP-E1 domain and identified the previously described loop region of the growth factor like domain  to be es


A crystal structure reveals an elegant mechanistic switch whereby helical bending and catalytic domain rotation allow self-activation of a histidine kinase during a bacterial stress response. Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from  S. mutans. VicK is composed of several domains  in addition to a short transmembrane domain. We find that the dimeric VicK protein has an elegant rod-shaped structure with the domains linearly connected like beads on a string. The structure suggests that VicK kinase activates itself by helical bending of the DHp domain and coordinated swinging around of the catalytic CA domain to engage with the target histidine. Structure-based mutagenesis experiments also helped us to identify key residues that are required for VicK's opposing phosphatase activity. Our studies of the multi-modular VicK protein suggest a sequential kinase activation model that may involve helical bending of the DHp domain and repositioning of the CA domains.Two-component signal transduction systems (TCSs) are promising targets for new antimicrobial research because they help bacteria and fungi adapt and survive. One of the main components of TCSs is a sensor histidine kinase (SK), which relays extracellular signals to intracellular pathways. Despite intensive research, a full-length structure of an SK has yet to be solved. In this study, we report the first crystal structure of the complete cytoplasmic region of VicK, an important SK in the tooth decay pathogen  Protein phosphorylation is an essential signal carrier. Bacteria respond to transient living environments through transmembrane-integrated sensor histidine kinases (SKs), which act in concert with their intracellular cognate response regulators (RRs) to elicit necessary adaptive responses that are critical for their survival and virulence. The SKs and RRs have evolved into a two-component signal transduction system (TCS), whereby stimulation of the SK autophosphorylates at a conserved histidine residue to initiate a signaling cascade Drosophila) are sensors in a majority of SKs, which respond to alterations in the redox potential, oxygen content, light, and small molecules in their environments Sensor domains are key modulators for SKs Archaeoglobus fulgidus revealed an unusual knobs-to-knobs interhelical structure, which suggests a coordinated helical rotation model for the HAMP domain in signal transmission The enzymatic activities of SKs are modulated by HAMP domains, which are commonly found in histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis, and phosphatase proteins Bacillus subtilisThe C-terminal catalytic and ATP-binding domain (CA) of the SKs, also called HATPase_c, in addition to the DHp domain (dimerization and histidine phosphorylation domain), phosphorylates a conserved histidine residue in the middle of DHp helices Streptococci, Bacilli, and StaphylococciS. mutans, which is an important pathogen in caries etiology, regulates acid production and tolerance conducive to dental caries, including proton expulsion (F1F0-ATPase) VicRK is a well-characterized TCS that is highly conserved and essential for survival and virulence in a wide range of firmicute bacteria, including Given the vital role of TCSs in bacterial adaptation and pathogenicity, decades of research have focused on the molecular mechanisms of TCS signaling cascades S. mutans VicK has one transmembrane domain  that anchors itself to the cytoplasmic membrane  and both C-terminal tails  are disordered.VicK crystals diffracted slightly better than 3 Å; however, because of strong anisotropy, the final structure was refined up to only 3.3 Å . The ove patches . In addi patches . The C-t patches . The N-tS. mutans HAMP shares approximately 45% identity to other Streptococci; however, it shows little (∼5%) identity to A. fulgidus Af1503, although the critical residues at positions a and d are mostly conserved (The HAMP domain (aa 36–86) is located at the uppermost position within the N-terminal region of the VicK structure . Helicesonserved .b, e, and g of the coiled-coil in addition to two residues from loop L1  in VicK, which is located downstream of the HAMP domain, adopts a canonical fold . Both PAS. pneumoniaeThe VicK PAS domains form a unique dimer, which is mediated by a leucine-zipper . This leTo look for clues of ligands for VicK PAS domains, we compared the VicK PAS domains with representative structures of all ligand-bound PAS domains according to a recent comprehensive review by Henry and Crosson The C-terminal end of VicK (aa 199–450) contains a histidine-specific ATPase, which is often divided into one DHp domain and one CA domain with a short linker (aa 270–278) . The dimBoth CA domains (aa 278–450) of VicK adopt a classical histidine kinase fold and are well aligned with an rmsd of 1.3 Å despite the missing loop L11 . The CA Unlike the HAMP domain, the DHp domain forms an anti-parallel four-helical coiled-coil. Interestingly, the two monomers of the DHp domain bear an asymmetric fold . The twoT. maritima HK853, which is a symmetric four-helix coiled-coil, we observed similar bending of monomer A , which resulted in a mobility retardation shift  to detect the phosphorylated on shift , lane 2.We tested the phosphatase activity of the DHp mutants described above. To our surprise, the single proline or threonine mutations (P222A and T221A) abolished VicK phosphatase activity .S. pneumoniae VicR eluted at ∼7.6 min compared with the native VicR at ∼8.8 min . Phosphorylated ∼8.8 min , run 1. ∼8.8 min , run 2. ∼8.8 min , run 3. Together, our data suggest that Pro222 and its neighbor, Thr221, are the key residues in the bending region required for the phosphatase activity of VicK.T. maritima HK853, we found that monomer A takes completely different positions  that disrupts both groups of key residues completely eliminated the autokinase activity , which comprises an SK dimer with one signal transducer, one signal sensor, and one intact kinase domain. This structure allows us to dissect the molecular mechanisms underlying SK-mediated signal transduction in prokaryotes.Our crystal structure revealed a long rod-shaped VicK holoenzyme . The thrB. subtilis, YycG proteins center in the division ring possibly through interactions with FtsZ, a tubulin-like protein S. pneumoniae cell The long shaped molecules often have the largest radius of gyration and tend to polymerize. VicK and its homologs have one or two integrated TMs rather than being free cytosolic proteins and are localized as clusters within the membrane Pseudomonas aeruginosa Aer-2 implicates that the conformational dynamics may also serve a role in signal transduction A long-standing question is how HAMP domains serve in signal transduction because they often directly connect TMs and extracellular sensors. The HAMP domains may undergo a 26° rotation, which is derived from the unusual knobs-to-knobs packing of the solution structure of Af1503 HAMP, when they receive transmembrane signals The VicK HAMP domain appears to be a stable four-helix coiled-coil with classical knobs-into-holes interactions, wherein three pairs of hydrophobic residues from helix α2 form a central hydrophobic core with knobs-to-knobs packing, and it is unlikely that structural alternations of the HAMP domain play a central role in signal transmission  and S1B.It is worth noting that the α2 helices of the HAMP domain connect with downstream PAS domains through continuous helices, part of which (aa 86–103) form a fairly rigid leucine-zipper . This loS. pneumoniae VicK reduced the autokinase activity and, more dramatically, phosphatase activity T. maritima ThkA S. mutans VicK structure shows that Leu135  stabilizes helix α4 by interactions with Ile116 and Ile139 , which contributes to the hydrophobic cavity of the PAS domain (unpublished data). Therefore, it is likely that an Ile139 to arginine mutation only disrupted potential ligand binding but not dimerization. In contrast, an L100R mutation disrupted dimerization of the PAS domain, and in turn, the kinase activity of S. pneumoniae VicK The VicK PAS domains form a stable dimer through the short leucine-zipper (aa 89–103) . In geneIn ligand-free VicK, helix α5 adapts an open conformation when compared to flavin adenine dinucleotide (FAD)-bound NifL, heme-bound FixL, and malate-bound DcuS . Thus, lB. subtilis DesK bends 50–54° when His188 is phosphorylated The local region around the phosphoryl receptor histidine of DHp domains is highly conserved Residues Pro222 and Thr221 are conserved among VicK homologs . InteresB. subtilis DesK has a glycine instead of proline present in this region B. subtilis Spo0B and Spo0F complex compared with T. maritima HK853 and RR468 complex, although a glycine residue is localized adjacent to the phosphoryl receptor histidine Glycine may also serve a similar role as proline. The glycine in the middle of an α-helix attributes unique flexibility A positions toward to its own His217 to form an active state, which is consistent with the findings using heterodimeric kinase mutants of HK853 and PhoR B. subtilis DesK have shown that the CA domains could position themselves differently relative to the DHp domain Our structure has shown that monomer A is 1140 Å2. However, this interface is mainly composed of hydrophilic and polar residues, which suggests that these contacts may only be transient (S. pneumoniae VicK completely abolished its autokinase activity and greatly reduced phosphatase activity The buried surface of the active monomer ransient . Our mutransient . Interesransient  and S10.ransient . The T22ransient  and 5. INb)  . Howeverin cis rotates ∼61° to reach this histidine for initial phosphorylation (II). It is likely that this activation does not happen simultaneously for both CA domains because the DHp domain allows only one helix to bend at a time  was cloned into vector pET15 (Novagen). S. mutans VicR (aa1–235) and S. pneumoniae VicR (aa1–234) were cloned into pETHis vector. All these constructs were expressed with N terminal 6× Histidine tag in Escherichia coli BL21/DE3 Rosetta (Novagen). VicK protein was purified through nickel affinity agarose (Qiagen), Q sepharose, phenyl sepharose, and Superdex S200 . The two VicR homologs were simply purified by nickel affinity agarose and gel filtration. Protein preps were concentrated down to 8 mg/ml in a buffer of 10 mM Tris (pH 8.0), 100 mM NaCl, 300 mM ammonium acetate, 2 mM DTT, and 1 mM β-mecaptoethanol (β-ME). The preliminary crystals were obtained using the screen kits of JCSG Plus (Qiagen) and Index (Hampton Research). Most crystals from these initial screening could only diffract to ∼4.5 Å. The later optimizations of those crystals defined the best condition of 2.3–2.8 M Sodium formate, 50 mM Tris (pH 7.4–8.6), and 4% PEG 4000 at room temperature. Selenium-methionine labeled protein was prepared following a standard protocol Bar-like crystals were grown for 2–4 d and immediately frozen in liquid nitrogen after quickly soaked with crystal growth buffer and additional 12% isopropanol as a cryo-protectant. Data were collected in BM17U, Shanghai Synchrotron Radiation facility (SSRF), China and processed by HKL2000 The selenium sites were located using SHELXD Model analyses were performed using a variety of programs. The structural alignments were calculated in Coot 2SO4 at 25°C. Data were then collected on a DAWN HELEOS II laser photometer with an emission at 658 nm . Molecular mass was calculated using ASTRA V .The VicK protein prep at ∼4 mg/ml was first resolved on a size exclusion column (Shodex KW-802.5) in a buffer of 50 mM HEPES (pH 7.0) and 200 mM Na32P-ATP  according to a recent protocol published by Winkler and his colleague 2 for 0.25 to 8 min at room temperature. The measurements were setup at four different time points. The protein concentration and time points for each VicK protein were pre-determined to have the signals within linear ranges. The reactions were stopped by adding 3 µl 4× SDS loading buffer. The resulted mixtures were then subjected to 12% SDS-PAGE before the gels were dried and scanned in Typhoon 9410 . Quantifications were carried out using a program Totallab Quant. The autokinase parameters were derived from non-linear regression of the Michaelis-Menten equation. For simple comparison of the various VicK mutants, the VicK protein preps of 2 µg were mixed with 0.3 µl hot ATP working solution in 15 µl reaction buffer and further incubated for 30 min at room temperature. The reactions were stopped with 4× SDS loading buffer and separated in 15% SDS-PAGE. The gels were dried before being exposed to X-ray film.All VicK mutants were generated by our modified Quikchange mutagenesis protocol S. pneumoniae VicR at concentration of 4 µM was phosphorylated in 50 mM AcP, 50 mM Tris (pH 7.4), 50 mM KCl, 2 mM MgCl2, and 20% glycerol for 1 h at 37°C 2. The gels were run at 120 V for 120 min at 4°C for the best mobility shift and stained with coomassie blue.The reactions of phosphorylated VicR were also analyzed by HPLC Homologs of the full-length VicK with >50% sequence identities were initially pooled from Genebank using a program BLAST The coordinates of the structure and relevant information have been deposited into the Protein Data Bank (4I5S).Figure S1Structural analyses of VicK HAMP domain. (A) Ribbon presentation of VicK HAMP domain. The right is the top view, 90° rotated along a horizontal axis of the dashed line. The colors and labels are the same as those in (JPG)Click here for additional data file.Figure S2The canonical PAS domain and its interaction network with leucine zipper. (A) Alignment of two canonical PAS domains of VicK. The color scheme is the same as that of (JPG)Click here for additional data file.Figure S3Sequence alignment of ligand-bound PAS domains with that of VicK. All representative ligand-bound PAS domains were selected according to a recent review by Henry and Crosson (TIFF)Click here for additional data file.Figure S4Structural alignments of ligand-bound PAS domains with S. mutans VicK. The alignments were performed by Coot Azotobacter vinelandii NifL  Bradyrhizobium japonicum FixL  E. coli DcuS  Salmonella typhimurium PhoQ  (JPG)Click here for additional data file.Figure S5Structure of the VicK C terminal DHp/CA domain. (A) C terminal domains of VicK shown in ribbon. The color scheme is the same as that described for T. maritima HK853 aligned with both VicK CA domains. HK853 CA domain is colored in grey and VicK CA domains in magenta and gold. ADP is taken from the HK853 structure.(JPG)Click here for additional data file.Figure S6Alignment of the DHp domain of the non-redundant VicK homologs. The alignment is colored by default in CLUSTAL program Streptococcus; E, Enterococcus; L, Lactobacillus; Lc, Lactococcus; P, Pediococcus. The alignment of all full-length homologs is presented in (JPG)Click here for additional data file.Figure S7Mutations in DHp domain did not affect the interaction between VicK and VicR.  The interactions of VicK and VicR were detected by native PAGE. All buffers were essentially the same as regular SDS-PAGE but without SDS. The 10% gels were used and run under 120 V for 120 min at 4°C and stained with coomassie blue. VicR, VicK, and the VicK/VicR complex are indicated by black, green, and red arrows, respectively. The wt VicK and mutants are labeled on top of each gel.(JPG)Click here for additional data file.Figure S8Alignment of the C terminal VicK with HK853. The alignment was performed as described in T. maritima HK853 in blue and cyan. The red circle is an axis in parallel to the DHp domain and going to the paper. The black dashed line is an axis perpendicular to the DHp domain. Grey arrows indicate rotation directions and rotation angles are labeled. His217 is highlighted in green sticks.(JPG)Click here for additional data file.Figure S9Interface between the DHp and active CA domain. The CA domain is presented in an electrostatic potential surface as described in (JPG)Click here for additional data file.Figure S10Detailed interactions between the VicK DHp domain and inactive CA domain. Residues involved in hydrogen bonds and hydrophobic interactions are labeled in sticks. Hydrogen bonds are shown in yellow dashed lines.(JPG)Click here for additional data file.Figure S11Alignment ofS. mutansVicK homologs. The alignment are colored by default in CLUSTAL program (PDF)Click here for additional data file.Figure S12Crystal contacts of two VicK dimers in one asymmetric unit. The top dimer is in ribbon colored and labeled as described in (JPG)Click here for additional data file.Table S1Autokinase activity of VicK wt and mutants. *The measurement performed at a protein concentration of 0.5 µM with time points of 15 s, 30 s, 45 s, and 60 s. **Not able to determine. ***The measurement performed at a protein concentration of 1 µM with time points of 15 s, 30 s, 45 s, and 60 s. #The measurement performed at a protein concentration of 10 µM with time points of 60 s, 120 s, 240 s, and 480 s.(XLS)Click here for additional data file.Table S2Statistics of data collection and structure refinement.†The data for the highest resolution shell are shown in parenthesis. *Rsym = ∑|I−<I>|/∑I, where I is the observed intensity, <I> is the statistically weighted average intensity of multiple observations of symmetry-related reflections. ††I/σ(I) – ratio of mean intensity to a mean standard deviation of intensity. ‡R = ∑||Fo|−|Fc||/∑|Fo|, where Fo and Fc are observed and calculated structure factor amplitudes, respectively. **Rfree is calculated using 5% of randomly selected reflections. ***Number of protein atoms of the ordered regions. §Rmsd – root mean square deviation.(DOC)Click here for additional data file.


Here we report crystal structures of the mouse PARG (mPARG) catalytic domain, its complexes with ADP-ribose (ADPr) and a PARG inhibitor ADP-HPD, as well as four PARG catalytic residues mutants. With these structures and biochemical analysis of 20 mPARG mutants, we provide a structural basis for understanding how the PAR polymer is recognized and hydrolyzed by mPARG. The structures and activity complementation experiment also suggest how the N-terminal flexible peptide preceding the PARG catalytic domain may regulate the enzymatic activity of PARG. This study contributes to our understanding of PARG catalytic and regulatory mechanisms as well as the rational design of PARG inhibitors.Protein poly(ADP-ribosyl)ation (PARylation) regulates a number of important cellular processes. Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for hydrolyzing the poly(ADP-ribose) (PAR) polymer  Reversal of PARylation is predominantly carried out by poly(ADP-ribose) glycohydrolase (PARG) in nucleus and cytosol, whereas ADP-ribosylhydrolase 3 (ARH3) may play a role in mitochondrial PAR degradation Protein function and localization inside the cell are usually regulated by post-translational modifications (PTMs). Poly(ADP-ribosyl)ation (PARylation) is a reversible PTM that is involved in various cellular processes, including DNA repair, chromatin structure dynamics, gene transcription, poly(ADP-ribose) (PAR) dependent cell death (pathanatos) and PARylation dependent ubiquitination PARG gene encoding for at least three different isoforms of PARG localizing in different cellular compartments. The 111 kDa full length PARG (hPARG111) localizes in the nucleus. Both 99 kDa hPARG99 and 102 kDa hPARG102 isoforms localize in the cytoplasm. While the N-terminal region is absent in some PARG splicing forms and predicted to be disordered  in mouse showed impaired DNA repair response with high genomic instability, including chromosome aberrations and a high frequency of sister chromatid exchange PARG activity is essential for many cell types. Loss of PARG function in M≈10 µM) than long and linear polymers (KM = 0.1–0.4 µM) It has been reported that vertebrate PARG possesses both exo-glycosidase and endo-glycosidase activities and therefore is able to hydrolyze ribose-ribose glycosidic bonds between ADP-ribose units at the terminus or within the PAR polymers 50 of about 120 nM. ADP-HDP has been used for in vitro studies for PARG inhibition. However, it is not cell permeable and can be hydrolyzed by phosphodiesterases in the cell, which make it unsuitable for cell based studies. The lack of an ideal small compound inhibitor for PARG is still a major hurdle for function studies of PARG. Recently, inhibitors of PARG have been proposed as drug targets in pathophysiological conditions such as inflammation, ischemia, and stroke Among proposed PARG inhibitors, adenosine 5′-diphosphate-(hydroxymethyl)-pyrrolidinediol (ADP-HPD), an analogue of ADPr, is probably the most potent and best studied one, with an ICT. curvata PARG, and the PARG catalytic domains of protozoan T. thermophila, rat and human were reported iso-ADP-ribose, which is probably the smallest PARG subtrate containing the α(1→2) ribose-ribose glycosidic bond, which may explain the processivity of PARG activity. Furthermore, through a complementation experiment, we show that the N-terminal regulatory fragment can activate in trans the inactive PARG fragment depleted with this segment. This suggests that, whereas the PARG activity can be inhibited by disrupting the docking of this segment to its PARG binding groove , PARG can be reversibly activated once the disruptive factor is removed. Altogether, our crystallographic and biochemical studies provided further insights into the catalytic and regulatory mechanism of mamalian PARG.To understand how PARG catalyzes PAR degradation and how it is regulated, and to provide a structural basis for PARG inhibitor development, we have independently determined crystal structures of a mouse PARG fragment roughly corresponding to the fully-active 60 kD fragment, in apo-form, and in complexes with ADPr or a PARG inhibitor ADP-HPD. Our apo-mPARG structure was one of the first released eukaryotic PARG structures (PDB ID: 4FC2). During our manuscript preparation, crystal structures of the bacterial PARG comprises an N-terminal regulatory/targeting domain and a C-terminal catalytic domain. The N-terminal region of mouse PARG (1–438) is absent in some PARG splicing forms, and is predicted to be disordered as shown by the metaPrDOS server  [30]. InThe mouse PARG catalytic domain has a bean-shaped structure, with the active site in a deep cleft in the middle on the abdominal side. A nine-strand mixed β sheet is sandwiched by two helical domains. The N-terminal helical domain has nine α helices, whereas the C-terminal helical domain has five. The very N-terminal segment of the mPARG catalytic domain contains the sixteen-residue putative mitochondrial targeting sequence  and binds to a groove on the side opposite to the active site . The cor6-8-QEE PARG signature catalytic loop (loop 1), the di-phosphate binding loop (loop 2) that is highly conserved among PARGs and other macrodomain structures There are three loops in the PARG catalytic cleft: the GGG-X1212) successfully soaked in ADPr and ADP-HPD, and solved the complex structures by molecular replacement , both methods failed, since the active site cleft is close to another copy of the mPARG molecule in the crystal lattice. Under a new crystallization condition, we obtained a second crystal form  encoded segment may serve as a reversible PARG activity switch (see discussion).To further investigate how this N-terminal extended region affects the PARG activity, we purified GST-tagged exon 4 coded peptide (GST-Exon-4) and GST-tagged exons 4 and 5 coded peptide (GST-Exon-4+5), and PARG catalytic domain exon 4 coded region deleted (ΔExon-4) and exons 4 and 5 coded region deleted (ΔExon-4+5). We ran the PARG TLC assay to study whether these N-terminal peptides can restore the PARG activity of these inactive PARG fragments in trans . Our dat6-8-QEE). A putative oxocarbenium intermediate is formed by the protonation of the (n-1) ADPr adenine-linked ribose 2′-OH leaving group through Glu. This positively charged oxocarbenium is stabilized by the proximal diphosphate group of bound ADPr. A water molecule is positioned to attack the oxocarbenium intermediate, which is activated through deprotonation by Glu. This results in the release of ADP-β-ribose and (n-1) PAR Based on the bacterial, protozoan, rat and human PARG structures, the mechanism of PARG catalysis was proposed to necessitate the binding of the terminal ADPr unit which in turn, positions the ribose-ribose O-glycosidic bond in direct hydrogen bonding contact with the last Glu residue of the signature catalytic loop . Then shorter PAR leaves PARG, and is degraded by PARG distributively  ADPr . Thus thbutively . It has While the catalytic mechanism of vertebrate PARG is better studied, little is known about the regulatory mechanism of PARG activity. One structural component known to be crucial for PARG activity is the Exon 4–5 encoded region. Removal of either exon would abolish PARG activity The exon4+5 region contains ∼70 residues. The high conservation of this region among vertebrate PARGs  suggests an important regulatory role it may have for vertebrate PARG activities. Interestingly, in our structure and two other vertebrate PARGs, this exon 4 and 5 encoded region has relatively high B factors  than the macro-domain like region of PARG . We propIn summary, through the determination of high resolution structures of mPARG and PARG mutants, in both apo- and liganded states, and enzymatic assays of mPARG mutants, we provide a basis for understanding the catalytic mechanism for mouse PARG. Our structures and the activity complementation experiment also suggest a model for PARG regulation. All these works will be valuable for understanding the molecular mechanisms of PARG in cell regulation and for PARG inhibitor development.E. coli BL21 (DE3) cells (Novagen) grown in Luria broth media. Se-Met substituted GST-mPARG(439–959) was over-expressed in auto-induction media. Bacteria cell pellets were lysed by sonication. Both native and Se-Met GST fusion proteins were eluted from Glutathione Sepharose 4B beads. GST tag was removed by TEV at 4°C overnight. Then the proteins were further purified by an anion exchange column, and finally purified by a Superdex 200 column on FPLC . The peak fractions were pooled, and concentrated to ∼5 mg/ml in a buffer containing 10 mM Tris HCl pH 8.5, 100 mM NaCl, 2 mM DTT. The mutants of mPARG(439–959) were cloned by site-directed mutagenesis. The mutant proteins were expressed and purified using the same methods as for the wild type protein.The gene fragment corresponding to mPARG catalytic domain (residues 439–959) was cloned into pGEX-4T1 with an N-terminal GST tag and a TEV cleavage site in-between. Native GST fusion protein was over-expressed in 4)2SO4 at 4°C. The best crystals were obtained by further micro-seeding in 14% PEG3350, 0.2 M (NH4)2SO4. Thick plate-shaped crystals usually appeared in one day at 4°C after seeding and grew to their full sizes in three days. The crystals were frozen by liquid nitrogen in the cryo solution containing 10% glycerol and 20% PEG3350. For the crystals of ADPr and ADP-HPD bound structures, 1 µL of protein sample with 25% glycerol was mixed with 1 µL of well solution containing 0.22 M KI, 20% PEG3350, 10 mM DTT at room temperature. The crystals grew to full size in 2–3 days, and were soaked with 1 mM ADPr or ADP-HPD overnight at room temperature. Then they were frozen by liquid nitrogen in cryo solution containing 10% glycerol and 20% PEG3350.The hanging-drop vapor diffusion method for crystallization was used to prepare crystals of the Se-Met mPARG(439–959). To obtain protein crystals for structural studies of unliganded and mutants mPARG E748N, E749N, E748Q and E749Q, 1 µL of protein sample (5 mg/mL) was mixed with 1 µL of well solution containing 20% PEG3350, 0.2 M (NHScreening and data collection were performed at the Advanced Light Source (ALS), beamlines 8.2.1 and 8.2.2 at wavelength 0.9793 Å. All diffraction data were processed by HKL2000 32P-labeled automodified PARP-1 was synthesized essentially as described by Ménard and Poirier 2, 8 mM dithiothreitol (DTT), 10% (v/v) glycerol, 25 µg/ml calf thymus activated DNA, 1 mM NAD+ and 125 µCi of 32P-NAD+]. Ethanol was added to this preparation dropwise at 10% (v/v) final concentration, with constant mixing, and the reaction mixture was incubated for 3 min at 30°C. The reaction was started by adding 20 units of PARP-1 purified up to the DNA–cellulose step (600 units/mg of protein) as described by Zahradka and Ebisuzaki et al.32P-labeled automodified PARP-1. pADPr hydrolysis was started by the addition of PARG mutants to a final concentration of 0.1 µM. Samples were incubated at 30°C for the indicated times. PARG activity was measured by analysing the production of ADP-ribose monomers from automodified PARP-1. PEI-F (polyethyleneimine F) cellulose (Macherey-Nagel) TLC developed in 0.3 M LiCl and 0.9 M acetic acid according to Ménard and Poirier PARG assays were performed in a final volume of 20 µl containing 20 mM potassium phosphate (pH 7.2), 50 mM KCl, 0.1 mg/ml BSA, 0.1% Triton X-100, 10 mM DTT and 20 µM of GST, GST-Exon-4 or GST-Exon-4+5 were pre-incubated with PARG ΔExon-4 or ΔExon-4+5 at different molar ratio. Then the PARG activity assay was performed as above, and the result was analyzed by TLC.Proteins were resolved using 4–12% Criterion™ XT Bis-Tris gradient gel (Bio-Rad) and stained with Sypro Ruby (Bio-Rad) according to the manufacturer's instructions. Images were acquired using the Geliance CCD-based bioimaging system (PerkinElmer).Protein Data Bank: Diffraction data and coordinates of mouse PARG catalytic domain are deposited under accession codes 4FC2, 4NA0, 4NA4, 4NA5, 4NA6, 4N9Y and 4N9Z, respectively.Figure S1The disorder prediction for mouse PARG from metaPrDOS server. The X-axis corresponds to mouse PARG residue numbers 1–969. The Y axis is the disorder tendency for each residue. The blue curve is the average result from six different programs/servers, as summarized by the metaPRDOS server. Higher values indicate higher disorder propensity. It indicates the N-terminal regulatory domain of mPARG (1–438) is disordered, whereas the mPARG(439–959) protein that was used for crystallization trials was predicted to be well-folded.(PDF)Click here for additional data file.Figure S2The core structure of mPARG has a macrodomain-like fold. The macrodomain-like region is highlighted in pink. mPARG has more delicate structure than macrodomain, including the N-terminal extended loop, seven more helices in the N-terminal helix bundle, two more helices in the C-terminal helix bundle, and three more N-terminal β strands . In addition, mPARG has an additional segment that contains the “Tyr” clasp (highlighted in red) within the macrodomain-like region.(PDF)Click here for additional data file.Figure S3Coomassie Blue Stained SDS-PAGE for the purified recombinant mPARG(439–959) wild type and mutants. The red asterisk indicates the expected position for mPARG(439–959).(PDF)Click here for additional data file.Figure S4A potential secondary iso-ADPr binding site.(A) Stereoview of the iso-ADPr binding site. R478, D480, F491 and T493 are highlighted in pink sticks. These residues are highly conserved in vertebrate PARGs. Fo - Fc difference density (grey mesh) is calculated when iso-ADPr is omitted (contoured at 2.5 σ). iso-ADPr is also observed in both E748Q and E749Q mutants structures at the same site. E748Q is in p21212 space group (B), and E749Q is in p21 space group (C). Fo - Fc difference density (grey mesh) is calculated when iso-ADPr is omitted (contoured at 2.5 σ).(PDF)Click here for additional data file.Figure S5The 2′-OH group of the adenine-linked ribose is exposed to solvent. The surface of the mPARG is shown as grey. ADPr analog ADP-HPD is highlighted as sticks. Unlike bacterial PARG, mPARG does not block the 2′-OH of the adenine-linked ribose. This allows the binding of (n+1) ADPr unit. This structure feature supports that mPARG has both exo- and endo-glycohydrolase activity.(PDF)Click here for additional data file.Table S1Statistics for data collection and structure refinement of mouse PARG(439–959) crystals.(PDF)Click here for additional data file.


Thus, the C-terminal domain may play both a positive and negative role. Interestingly, a synthetic peptide corresponding to the full C-terminal domain (residues 576–615 of BAK1) interacted with recombinant BRI1 in vitro, and that interaction was enhanced by phosphorylation at the Tyr-610 site. Expression of a BAK1 C-terminal domain truncation (designated BAK1-ΔCT-Flag) in transgenic Arabidopsis plants lacking endogenous bak1 and its functional paralog, bkk1, produced plants that were wild type in appearance but much smaller than plants expressing full-length BAK1-Flag. The reduction in growth may be attributed to a partial inhibition of BR signaling in vivo as reflected in root growth assays but other factors are likely involved as well. Our working model is that in vivo, the inhibitory action of the C-terminal domain of BAK1 is relieved by binding to BRI1. However, that interaction is not essential for BR signaling, but other aspects of cellular signaling are impacted when the C-terminal domain is truncated and result in inhibition of growth. These results increase the molecular understanding of the C-terminal domain of BAK1 as a regulator of kinase activity that may serve as a model for other receptor kinases.Binding of brassinolide to the brassinosteroid-insenstive 1(BRI1) receptor kinase promotes interaction with its co-receptor, BRI1-associated receptor kinase 1 (BAK1). Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not entirely clear. In the present study we show that the carboxy (C)-terminal polypeptide of BAK1 may play a role. First, we demonstrate that the C-terminal domain is a strong inhibitor of the transphosphorylation activity of the recombinant BAK1 cytoplasmic domain protein. However, recombinant BAK1 lacking the C-terminal domain is unable to transactivate the peptide kinase activity of BRI1  Overall, the results establish an important role for the C-terminal domain of BAK1 and perhaps other receptor kinases as well.In the present study, we show that the C-terminal domain of BAK1 inhibits the transphosphorylation activity of BAK1 but is essential for BAK1 to activate BRI1 activity Arabidopsis thaliana ecotype Ws-2 was the background for the bri1-5 mutant and Col-0 was the background for transgenics in the null bak1-4 bkk1 mutant background. Expression of BAK1::BAK1-Flag (wild type and site-directed mutants) in the bak1-4 bkk1-1 double mutant background was achieved by transforming the viable heterozygous BAK1-4 /bkk1-1 bkk1-1  and 2% (w/v) sucrose, pH 5.7]. For phenotypic analysis between plants expressing BAK1-Flag and BAK1-ΔCT-Flag, plates containing half-strength MS medium (0.8% agar) were kept at 22°C with an 8-h photoperiod for 9 days. Seedlings were then transferred to soil and grown in the same temperature and light conditions to analyze phenotype.Seeds were surface sterilized, kept at 4°C for 2 days, and then sown on germination medium ; twice with PBS150 [5 mM NaP, 150 mM NaCl); once with PBS50 ; and finally two washes with IPDB solution. After the final wash step, an aliquot of 1.5X SDS buffer was added and the mixture was heated at 95°C for 5 min to elute BAK1-Flag and BAK1-ΔCT-Flag proteins. Recombinant Flag-BAK1 was immunopurified using anti-Flag M2 affinity gel  as previously described , anti-phosphothreonine antibodies (1:500 dilution), and anti-phosphotyrosine antibodies (1:500 dilution). Immunoblots were scanned using an Odyssey Infrared Imaging System  for visualization. Blots stained with ProQ Diamond phosphoprotein stain  were scanned using a Typhoon Molecular Dynamics phosphor/fluorescence imager.2 unless specified otherwise. Synthetic peptides corresponding to the C-terminal domain of BAK1 (residues 576–615) were produced with an N-terminal biotin group and either no additional modifications (CT), with phosphorylation of the Tyr-610 residue (CTpY610) or phosphorylation of the Ser-612 residue (CTpS612). All peptides were synthesized by GenScript Corp . Biotinylated peptides were captured to SA Streptavidin Biosensors followed by binding of the analyte Flag-BRI1 from a 100 μg/mL solution or as otherwise noted. One-shot KD was calculated based on the 1:1 model and the average KD was extracted from binding results for four different concentrations of the analyte.The real-time binding of Flag-BRI1  to immobilized BAK1 C-terminal domain peptides was monitored using an Octet QK . The binding buffer contained 50 mM MOPS (pH 7.5), 0.01% (v/v) Triton X-100, 20 mM NaCl and 5 mM MgClFigure 1A). Interestingly, only the constructs with the juxtamembrane domain (BAK1-JKC and BAK1-JK) had robust autophosphorylation on threonine and tyrosine residues . By monitoring the phosphorylation of E. coli proteins during production of the recombinant protein kinases . Collectively, these results suggest that the juxtamembrane domain is an activator of the kinase domain, both in terms of auto- and trans-phosphorylation activity, and that the C-terminal domain is a strong inhibitor in particular of transphosphorylation activity. Alternatively, the C-terminal domain may affect specificity of the BAK1 kinase domain such that additional bacterial proteins and phosphorylation motifs are recognized when the C-terminal domain is truncated, which could also result in increased transphosphorylation activity. It will be important in future studies to distinguish between increased activity per se and altered specificity.As an initial approach to study the role of flanking domains, we used the Flag-BAK1 cytoplasmic domain construct to produce a set of deletion mutants lacking the juxtamembrane (J) domain, the C-terminal domain, or both. All constructs had the kinase (K) domain with a Flag-epitope tag at the N-terminus . This was unexpected, because with the full-length BAK1 cytoplasmic domain protein, Tyr-610 in the C-terminal domain is a major site of autophosphorylation such that tyrosine autophosphorylation of the Y610F directed mutant is substantially reduced relative to the wild type protein . The surprising result was that the Y304F and Y363F directed mutants had substantially reduced tyrosine autophosphorylation because in the full-length cytoplasmic domain proteins these substitutions have relatively little effect on the overall level of tyrosine autophosphorylation , but whether that was the result of autophosphorylation of new sites or increased stoichiometry of known sites is not clear and was not explored in the present study.Another interesting point is that the BAK1-JK truncation had strong autophosphorylation on tyrosine residues . The full-length Flag-BAK1 cytoplasmic domain ends at residue 615 and only weakly transphosphorylates bacterial proteins on tyrosine and threonine relative to the BAK1-JK truncation mutant, which ends at residue 577. Removal of the last five residues of the C-terminal domain (to generate the CT610 mutant), substantially increased transphosphorylation activity on tyrosine and threonine residues, whereas the full effect required removal of the last twenty residues. Longer deletions beyond the CT595 truncation did not result in any further increase in transphosphorylation activity. These results suggest that the last half of the C-terminal domain is responsible for inhibition of transphosphorylation activity, possibly by interacting with the kinase domain and restricting protein substrate access to the active site.As an initial approach to determine which regions of the C-terminal domain are crucial to the transphosphorylation activity of BAK1, we constructed a nested series of truncations that sequentially removed five amino acid segments from the C-terminus of this flanking region  would have increased signaling and enhanced growth; however, that result was not obtained. As shown in Figure 4B, the BAK1-ΔCT-Flag plants had smaller roots when grown as seedlings on agar plates and the rosettes remained substantially smaller than plants expressing full-length BAK1-Flag during both vegetative and reproductive development . Expression levels of the BAK1-ΔCT protein varied among transgenic lines tested but were not responsible for the reduced growth phenotype . Moreover, both BAK1-Flag and BAK1-ΔCT-Flag were associated with BRI1 in vivo, as the two proteins were co-immunoprecipitated from all transgenic lines. Thus, the reduced growth was not the result of impaired binding of BAK1-ΔCT to BRI1 in vivo. Although growth of the BAK1-ΔCT-Flag plants was markedly reduced, rosette morphology was not indicative of impaired BR signaling as petioles and leaves were relatively elongated unlike strong BR signaling mutants  displayed peptide kinase activity. However, GST-BAK1 substantially increased the ability of Flag-BRI1to transphosphorylate the synthetic peptide substrate whereas the GST-BAK1-JK truncation mutant did not . As expected, the kinase-inactive GST-mBAK1 did not stimulate BRI1 peptide kinase activity because BAK1 kinase activity is required for the transphosphorylation/activation of BRI1  may reflect altered specificity (rather than increased activity) that allows the recombinant Flag-BAK1-JK protein to phosphorylate a broader array of bacterial proteins. If this broadened specificity occurred in planta, it may result in inappropriate signaling causing growth inhibition. We will test these possibilities in future experiments by analyzing gene expression in BAK1-ΔCT-Flag plants to ascertain whether defense genes are activated in the absence of (apparent) external triggers. We will also determine whether levels of FLS2 protein are reduced, as FLS2 degradation follows its activation . One possibility would be that when the two receptor kinases are juxtaposed, the C-terminal domain of BAK1 binds to BRI1, thereby helping to properly position the two kinase domains while also relieving the inhibitory effect of the C-terminal domain on BAK1 transphosphorylation activity. To test this notion, we asked whether recombinant Flag-BRI1 would bind to the immobilized C-terminal polypeptide of BAK1. As shown in Figure 7A, with a synthetic peptide based on the entire C-terminal domain of BAK1  as the immobilized ligand, recombinant Flag-BRI1 protein  interacted but only weakly. However, robust binding of Flag-BRI1 was observed with the C-terminal domain peptide phosphorylated at the Tyr-610 site . The increased binding could not be attributed simply to adding negative charge to the polypeptide because the CT peptide phosphorylated at the Ser-612 site (designated CTpS612) only supported slightly more binding than the unphosphorylated CT peptide . Binding of Flag-BRI1 to the CTpY610 peptide increased as pH of the reaction mixture was reduced from pH 8–7, but binding to the immobilized CT peptide was low and constant over the same pH range . Interestingly, early studies . It is recognized that many protein kinases, such as protein kinase A , which is similar to the affinity of the interaction between GST-BAK1 and Flag-BRI1 in vitro , which is also supported by the observation that plants do not have the morphology of BR signaling mutants . We speculate that other factors may be involved in reducing plant growth, such as enhanced stress signaling in plants expressing BAK1-ΔCT-Flag, perhaps as a result of FLS2 activation, or inappropriate signaling that occurs as a result of altered specificity of the BAK1-ΔCT-Flag protein. These are important questions but are beyond the scope of the present study. Much also remains to be done with the possible role of the C-terminal domain of BAK1 in its interaction with BRI1 resulting in BR signaling. In particular, it will be interesting to determine whether C-terminal domain binding between proteins applies to other receptor:co-receptor kinase pairs as well. Our present results are generally consistent with the previous report that C-terminal epitope tags can affect BAK1 function in different signaling pathways (Our results support a model that involves inhibition of BAK1 kinase activity by its C-terminal domain, which we postulate can be relieved by its binding to the cytoplasmic domain of BRI1 after formation of the BRI1:BAK1 complex. Interestingly, the flanking domains of BRI1 , 2012 anpathways . Our respathways , it willThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SAG), and lung glycoprotein-340 (DMBT1GP340) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1 InteracThe oral tissues, a part of the mucosal immune system, are constantly covered by saliva, which harbors a similar set of antimicrobial proteins as other mucosal fluids . A major2.SAG) [As a result of the continuous flow of saliva in our mouth, planktonic bacteria are swallowed, making microbial adherence one of the key selective criteria in the oral cavity ,8. SalivSAG) ,13.3.Streptococcus mutans is considered the major cause for dental caries. In search for S. mutans agglutinating substances, Ericson and Rundegren isolated a 300–400 kDa glycoprotein from parotid saliva [S. mutans. This protein was named salivary agglutinin (DMBT1SAG). It also binds a number of both Gram-positive and Gram-negative bacteria, as well as to host proteins including IgA, complement factor C1q, bovine and human lactoferrin, albumin and lysozyme [SAG is identical with lung glycoprotein 340 (DMBT1GP340) and Deleted in Malignant Brain Tumors-1 (DMBT1) [DMBT1 gene [SAG and DMBT1gp-340 cross react with monoclonal antibodies raised against the DMBT1s isolated from the different sources. Both proteins show calcium-dependent binding to S. mutans and SP-D [GP340 was purified from bronchoalveolar lavage fluid of patients with alveolar proteinosis [SAG and DMBT1GP340 thus represent DMBT1 isoforms that are encoded by the DMBT1 gene on chromosome 10q26.13. The DMBT1 gene shows frequent genomic rearrangements and/or loss of expression in two common types of malignant brain tumors, but also in diverse epithelial cancer types, including lung, gastric, esophageal, colon, breast, and skin cancer [d saliva  that sholysozyme –19. Seve (DMBT1) –22. The BT1 gene ,24. DMBTand SP-D . DMBT1GPteinosis . It bindteinosis , membersteinosis . DMBT1SAn cancer –36. A se4.N-terminal end of the DMBT1s, are short serine-threonine-rich amino acid motifs of 20–24 amino acids in length, called SRCR interspersed domains (SIDs). The largest naturally occurring allele known to date contains 13 of these SRCR-SID units, while the shortest known displays only eight. This genetic variability in normal individuals raises the question if size variations of DMBT1GP340, DMBT1SAG and variants from other tissues are due to alternative splicing and post-translational processing or are alternatively determined by genetic differences.The most typical feature of DMBT1 is that it consists of protein domains . DMBT1 iC-terminal end of the gene is a Zona Pellucida domain. Except for the SIDs, these domains are all known to be involved in ligand binding [The stretch of tandem repeated SRCR domains with their interspersed SIDs are followed by two CUB domains separated by a 14th SRCR domain, and at the  binding –39. DMBT5.Cryptosporidium, Toxoplasma and Plasmodium and algae of the genus Chlamidomonas [Strongylocentrotus purpuratus, which contains 218 genes comprising altogether 1,095 SRCR domains [The most prevalent domains in DMBT1 are the SRCR domains . The SRCidomonas ,44. The  domains . For the domains , SRCR do domains . The pos domains .6.Complement subcomponents (C1s/C1r), embryonic sea urchin protein , and bone morphogenetic protein 1 (Bmp1). CUB domains are found in numerous proteins that are mainly involved in developmental processes [The CUB domain is a 100–110 residue-spanning extracellular domain named after the three proteins, in which it was first recognized: rocesses . Almost rocesses .7.C-terminal of the polypeptide, which is also the case for DMBT1s. The functions of proteins containing a ZP domain vary tremendously. They are found in glycoproteins involved in development, acoustic perception, immunity, and cancer [SAG forms aggregates [The zona pellucida (ZP) domain was first recognized in the sperm receptor proteins ZP2 and ZP3 ,47. Thesd cancer . ZP domad cancer . This isgregates ,52.8.SAG accounts for 25–40% of the molecular weight [N-linked glycosylation sites [O-linked sites situated primarily in the SIDs. The SRCR domains, however, contain only few potential O-glycosylation sites (Ser and Thr residues). Presumably, the high density of glycans forces the SIDs in an extended conformation, as has been demonstrated for mucins [The carbohydrate part of DMBT1r weight ,52. DMBTon sites , and numr mucins , therebyO-linked structures in DMBT1SAG are extended core 1 and core 2 oligosaccharides, either neutral or monosialylated  [RVEVLYRGSW). Of these eight residues, four were always present in the binding motif (GRVEVLYRGSW). Of the 14 SRCR domains in the full length molecule, 12 contain the eight amino acids involved in binding giving bacterial binding of DMBT1 a multivalent character. One of the two deviating SRCR domains is the 14th SRCR domain (GRVEIYHGGTW), located between two CUB domains and as such probably not involved in bacterial binding.BT1pbs1) . AlthougRGRAEVYYSG) [SAG bacteria binding site were synthesized for all nine SRCR domains and tested for bacteria binding [SRCR domains in other proteins have previously been implicated in bacteria binding –70. The AEVYYSG) ,72. SimiAEVYYSG) ,74. CD 1 binding . One of 9.2.SAG-mediated agglutination of S.mutans is inhibited by high concentrations of fucose and lactose. In line with these results, it was shown that the Lea-antigen GlcNAc) was involved in S.mutans binding, harboring an essential role for the terminal fucose [SAG to S. sanguis and S. mutans [H. pylori can bind Leb structures and sialic acid on DMBT1SAG with its BabA and SabA adhesins, respectively [Studies of bacterial binding sites on SAG were first mainly focused on carbohydrate ligands, following the concept that bacteria primarily recognize carbohydrate ligands on mucosal surfaces ,77. Thesl fucose . Other s. mutans ,78. H. pectively .10.10.1.SAG was first described as an agglutinating agent for S.mutans and other streptococci [SAG on the dental surface promoted S. mutans colonization, but high concentrations in saliva inhibit its colonization [DMBT1ptococci . Severalptococci ,79–81. Hnization .SAG binds in a calcium-dependent manner to antigen I/II polypeptides, a group of surface receptors on S. mutans and related streptococci. Antigen I/II polypeptides have been characterized under different names in a variety of streptococci, S. mutans , S. sobrinus , S. gordonii , S. intermedius (Pas), S. pyogenes (aspA) and S. agalactiae (bspD) [C-terminal region of about 615 amino acids shows 64% or more sequence homology between the I/II polypeptides.DMBT1e (bspD) ,83. Antie (bspD) . These pSAG were identified on antigen I/II polypeptides and other proteins . DMBT1SAG binds to lipoteichoic acid, a component on the membrane of Gram-positive bacteria, and to lipopolysaccharide (LPS), a common component on the cell membrane of Gram-negative bacteria. LPS has been found to bind to SRCRP2, suggesting DMBT1 recognizes LPS with its SRCR domains [Dmbt1−/− mice were more susceptible to low doses of DSS than Dmbt1+/+ mice. However, since this is a commensal bacteria induced colitis model, this effect is likely to be attributed to Dmbt1’s ability to control the natural intestinal microflora.Bacteria that do not possess antigen I/II polypeptides, like DMBT1SAG ,65. CRP-eumoniae . As many domains . Binding11.SAG also binds viruses, including HIV-1 [SAG shows a calcium-dependent interaction with the virus gp120. The DMBT1SAG binding region on gp120 is localized to a linear, highly conserved sequence (CTRPNYNKRKR) near the stem of the V3 loop that is critical for chemokine receptor interaction during viral binding and infection [SAG most probably blocks the access of gp120 to the chemokine receptor. The N-terminal sequence of DMBT1, including the first SRCR domain and one-half of the first SID, binds in a Ca2+-dependent manner to the same HIV-1 V3 sequences previously identified to interact with full-length SAG [Besides bacteria, DMBT1ng HIV-1 ,103 and ng HIV-1 ,104. HIVng HIV-1 ,106. DMBnfection . Thus, Dngth SAG .SAG and DMBT1GP340 also inhibit the hemagglutination activity and infectivity of Influenza A virus (IAV) [SAG had significantly greater inhibitory activity against avian-like IAV strains which preferentially bound sialic acids in α linkage, than DMBT1SAG from another donor or three lung preparations of DMBT1GP340 [SAG from this donor as compared with the other samples. Hence, the specificity of sialic acid linkages on DMBT1 is an important determinant of anti-IAV activity. In addition to these direct virus-neutralizing properties, DMBT1GP340 displays cooperative interactions with SP-D in viral neutralization and aggregation assays. On the other hand, in some cases DMBT1SAG strongly antagonizes the antiviral activities of SP-D by binding to its carbohydrate recognition domain, which is involved in virus binding [DMBT1us (IAV) , which ius (IAV) . A variaBT1GP340 . In line binding .12.SAG binds to secretory IgA in a calcium-dependent manner, resulting in a cooperative effect on bacterial aggregation [SAG to IgA is mediated by the same 11-mer peptide that is responsible for the broad-spectrum bacteria binding of DMBT1SAG [SAG-IgA complex to the Pac molecule of S. mutans was demonstrated [SAG and concomitantly binding to Pac disappeared.DMBT1 binds to a number of endogenous proteins such as secretory IgA, MUC5B, surfactant proteins A and D, complement factor C1q, lactoferrin and albumin ,108,109.regation ,110. BinDMBT1SAG . Bindingnstrated . After cSAG also binds to C1q of the complement system, a system of plasma proteins that may react with one another to opsonize or permeabilize pathogens and induce a series of inflammatory responses [SAG is secreted in saliva and C1q is a serum component, these two fluids may mix in the oral cavity under conditions of oral inflammation, e.g., periodontal disease, or mucosal damage, thus enabling a local complement activation.DMBT1esponses . By bindesponses . AlthougPseudomonas biofilms [SAG binding to S. mutans protein antigen Pac, which belongs to the antigen I/II family [SAG that is responsible for S. mutans binding also mediates binding of lactoferrin [SAG binding. Although the homologous sequence in human lactoferrin is slightly different (SCKFDEYFSQSCA), human lactoferrin also binds to DMBT1 [DMBT1 also binds to bovine and human lactoferrin, an 80 kDa iron binding protein belonging to the transferrin family ,19. Lactbiofilms . In addibiofilms . Bovine I family . The SRCtoferrin ,89. BoviGP340, which originally was isolated as a soluble receptor for SP-D, also binds SP-A [N-terminal ends of the chains. Each polypeptide consists of a short N-terminal region, followed by a collagen-like sequence, a short α-helical sequence and the carbohydrate recognition domain. DMBT1GP340 binds to the carbohydrate recognition domain of SP-D through a calcium-dependent protein-protein interaction [DMBT1nds SP-A ,25. SP-Ands SP-A . SP-D foeraction . SP-A an13.Due to their broad pathogen binding spectrum, DMBT1 and its mouse homolog CRP-ductin are considered pattern recognition receptors (PRRs) , an anci14.Conclusively, DMBT1 plays a role in innate immunity by binding to a large panel of exogenous and endogenous ligands. SRCRP2 on the SRCR domains and sialic acids on the glycan chains have been identified as ligand binding sites, but DMBT1 may harbor many other potential ligand binding sites. These many ligand binding sites and its structural heterogeneity—both in the different tissues and between different individuals—are only two of the many surprises hidden in this molecule. Future research should focus on the precise role of DMBT1 in the innate immune system and the physiological consequences of its heterogeneity.