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Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA
(RECEIVED July 18, 2006; FINAL REVISION October 6, 2006; ACCEPTED October 13, 2006)
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Abstract |
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Keywords: neurophysin; ligand-facilitated dimerization; interdomain loop; amino terminus; subunit interface; hydrogen bonding; NMR; crystallography
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Neurophysins are a family of closely related proteins derived from either the oxytocin or vasopressin precursors (or analogous precursors in other species), and were among the first proteins for which ligand-facilitated dimerization was demonstrated (Nicolas et al. 1976, 1980). With one ambiguous exception, all ligands capable of binding to the neurophysin hormone-binding site—oxytocin, vasopressin, and related smaller peptides—similarly increase dimerization constants (Breslow et al. 1973, 1991; Fassina and Chaiken 1988). Given the importance of both hormone–NP interaction and NP dimerization to precursor stability (Barat et al. 2004), the linkage between binding and dimerization is likely to play a particularly significant role in folding in vivo.
The effect of ligand on NP dimerization is allosteric, as evidenced by the crystallographic demonstration of a clear separation between neurophysin's hormone-binding site and the dimer subunit interface (for example, see Rose et al. 1996; Wu et al. 2001). In a recent study of the weakly dimerizing H80E mutant of BNPI (bovine oxytocin-related NP), we presented evidence that dimerization of the unliganded protein is associated with changes at the hormone-binding site, supporting the view that differences in binding site conformation between unliganded monomeric and dimeric states, favoring binding to dimer, are significant contributors to the allosteric mechanism (Naik et al. 2005). The present study is aimed principally at exploring the contributions to ligand-facilitated dimerization of the neurophysin interdomain loop, a segment consisting of four or five residues that are distant from the dimer subunit interface, but that serve as a covalent bridge and potential hinge between the protein's amino and carboxyl domains. Because NP dimerization involves both domains, their relative orientation is important; changes in this orientation, controlled by an interdomain hinge, would have the ability to modulate dimerization (Eubanks et al. 2001; Nguyen and Breslow 2002).
We explored the role of the loop by examining the effects of loop residue mutation and of deletion of the six amino-terminal NP residues. Undefined pH-dependent intramolecular interactions of the first two residues of BNPII (bovine vasopressin-related NP) were first observed by 1D NMR (Lord and Breslow 1979) and subsequently shown to be released upon occupancy of the hormone-binding site (Zheng et al. 1997). Deletion of the first six residues of BNPII led to increases in dimerization and binding constants by factors of four and two, respectively, and, based on consideration of the crystal structure of WT liganded BNPII, it was suggested that the amino terminus might interact with the loop in the unliganded state (Zheng et al. 1997). In contrast, 1D NMR (Lord and Breslow 1979) did not indicate analogous interactions of the amino terminus of BNPI, the allosteric properties of which are similar, but possibly not identical, to those of BNPII (compare Kanmera and Chaiken 1985; Fassina and Chaiken 1988; Breslow et al. 1991). Thus, the potential importance of the amino terminus to NP allosteric effects was unclear.
More recently, however, NMR investigation of the structure of H80E (the H80E mutant of BNPI) demonstrated interactions of residues 3 and 4 with loop residue 55 in the unliganded monomeric state (Nguyen and Breslow 2002), an interaction now also seen by NMR in the unliganded WT BNPI dimer (H. Lee, M.T. Naik, C. Bracken, and E. Breslow, in prep.), suggesting that such interactions might modulate dimerization in both BNPI and BNPII, as we explore here. Interactions in the monomer NMR structure are also seen between the 1–6 sequence and both loop residue 56 and binding site residues 53 and 54, adjacent to the loop. Note that crystallography has provided little information about residues 1–6. These residues are largely undetectable (and assumed disordered) in complexes of full-length WT BNPII (Chen et al. 1991; Rose et al. 1996) and were deleted in other studies to improve data resolution (Wu et al. 2001).
Loop residues are strongly, perhaps completely, conserved among mammalian neurophysins (for example, see Chauvet et al. 1983). BNPI is chosen for these studies because of the body of preexisting NMR data on this system (for example, see Breslow et al. 1992; Nguyen and Breslow 2002; Naik et al. 2005) and our reliance on NMR to assess dimerization (Zheng et al. 1997). However, with the exception of the NMR structure of the unliganded H80E monomer (Nguyen and Breslow 2002), no BNPI structures have previously been solved. Instead, due to their easier crystallizability, BNPII crystal structures have provided the basis to date of structure-based insights into the effects of NP mutation. Crystal structures have respectively been solved for dimeric WT BNPII bound to the dipeptide FY and to oxytocin, and of des 1–6 BNPII both in the unliganded dimeric state and in the liganded dimeric state bound to vasopressin (Chen et al. 1991; Rose et al. 1996; Wu et al. 2001). Nonetheless, because BNPI and BNPII differ in 
20% of their sequences (Fig. 1) and no structure of dimeric BNPI has been available, and because of the effects of loop mutations reported below, the present investigation also includes elucidation of the structures of des 1–6 BNPI and its Q58V mutant in their liganded dimeric states, as well as the structure of desBNPI (as its F91STOP mutant) in its unliganded dimeric state. Discussion of these structures here will principally focus on features relevant to allosteric mechanism.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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50% (see Materials and Methods). In the case of the S56E mutant, the principal cause of the problem is a major reduction in peptide-binding affinity (Table 1), so that the folded state is not stabilized by bound ligand under folding conditions as normally (with only one known exception) required for efficient NP folding (for example, see Eubanks et al. 2000). However, because both the binding and dimerization properties of the folded K59G mutant were not significantly weaker than those of many mutants that did fold normally (Table 1)—particularly those of the closely related K59A mutant—and because neither CD nor NMR indicated any significant conformational differences from normally folded protein, a folding pathway defect was suspected. Consistent with this, changing the folding procedure (see Materials and Methods) from the 
-mercaptoethanol procedure in which the thiol catalyzing disulfide rearrangement is air-oxidized during folding, to the glutathione buffer procedure, which does not involve thiol oxidation and thereby allows a longer time for rearrangement of protein disulfides, resulted in normal folding efficiency. A potential explanation of the pathway defect is provided in the Discussion.
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Note in Table 2 that the thermodynamic effects of the Q58V, Q55A, and des 1–6 mutations exhibit variable additivity with each other and that the degree of additivity is not necessarily the same in a given double mutant for both binding and dimerization. For example, the effects on binding of the Q58V and Q55A mutations are almost completely additive, but their combined effects on dimerization exhibit extreme nonadditivity. The des 1–6 mutation is completely additive with the Q58V mutation when binding is measured, but is markedly nonadditive with the Q55A mutation, while slightly potentiating the effects of both these mutations on dimerization.
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Crystal structures of the FY complex of desBNPI and of unliganded desBNPIF91STOP
The BNPI crystals most suitable for X-ray diffraction analysis were those of its des 1–6 derivative in the liganded state and its des 1–6 F91STOP mutant in the unliganded state. Phe91 is close to the carboxyl terminus (Fig. 1); the F91 STOP mutation increased the dimerization constant by 50%–90% when either the full-length or des 1–6 derivatives were compared, but was otherwise benign (Table 1). (The dimerization increase is of undetermined origin since, as with BNPII, residues beyond 86 or 87 are not seen in the crystal structures.) Figure 5 shows the crystal structure of the asymmetric unit of desBNPIfy. Like the crystal structure of BNPIIfy (and unlike complexes of BNPII with hormone), the asymmetric unit of the unit cell contains more than one dimer. In the case of BNPIIfy, the two dimers interact directly via weak interactions (Chen et al. 1991). In desBNPIfy, the two dimers (AB and CD) are linked together indirectly by a single orthogonally bridging dimer subunit (E) from an adjacent asymmetric unit, with contacts to E involving subunits A and D. The E subunit appears to be a crystal phenomenon and is not considered in our analyses. However, despite the crystal packing and sequence differences between BNPI and BNPII (Fig. 1) and despite the fact that residues 1–6 are absent in the BNPI structure, the conformations of the individual chains of desBNPIfy and BNPIIfy are basically similar, as shown by a comparison of their backbones (Fig. 6).
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Residues directly participating in dimerization in both complexes are also, with one significant exception, the same in sequence position, albeit not necessarily in identity (Fig. 1)—principally 32–38, 40, 72, and 77–81—and show similar modes of interaction in the two neurophysins. The significant exception, which remains true on comparison of desBNPIfy with all BNPII complexes of known structure, lies in the finding that Ser25 forms a hydrogen bond across the interface only in the BNPI complex, an apparent consequence of the difference between BNPI and BNPII in interface residues 77 and 81 (Fig. 1). In both liganded and unliganded BNPI and BNPII dimers, the backbone –NH of 81 is hydrogen bonded across the interface to the carbonyl oxygen of 77 of the partner subunit. However, an additional intersubunit hydrogen bond is present in the liganded state that in both cases involves the residue 81 side chain.
In BNPII complexes, the –OH of Thr81 hydrogen bonds to the carbonyl oxygen of 77 of its partner subunit, representing a second hydrogen bond to this oxygen (Wu et al. 2001). The same bond cannot form at neutral pH to the side chain of Glu81 of BNPI. Instead, in three of the four chains of the two complete desBNPIfy dimers, the carboxyl group of 81 hydrogen bonds across the interface to the –OH of Ser25 (Fig. 7). The absence of this hydrogen bond in unliganded desBNPI is evidenced by a lack of visible electron density of the residue 81 side chain in any of the four dimer subunits, indicating disorder, as well as by structure refinement. RMSD comparison of the subunit interfaces of liganded and unliganded states indicates that formation of this hydrogen bond represents the largest ligand-induced interface change. For example, after structure refinement, the average RMSD between liganded and unliganded states of the side chains of all interface residues in the AB dimer, exclusive of residue 81, is 0.8 ± 0.4 Å, while that for residue 81 is 2.7 ± 0.3 Å. Amino domain interface changes are particularly small.
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Crystal structure of the dipeptide complex of the Q58V mutant of des 1–6 BNPI
Despite the effects on binding and dimerization of the Q58V mutation, the crystal structure of the dipeptide complex of desQ58V (including the arrangement of subunits within the asymmetric unit) is very similar to that of the corresponding des1–6 WT protein, as particularly exemplified by RMSD comparison of the structures of their respective A subunits (Fig. 8). Although additional residues show high RMSD values when other subunits are compared, almost all such residues (including those in subunit A), with the exception of the 77–81 region, represent solvent-exposed residues of no known function in the folded protein. In the rare instances where binding site residues are involved, deviations from the WT protein appear unlikely to contribute significantly to the higher binding constant of the mutant. However, differences in side chain orientation for residues 77 and 81, which interact with each other across the interface via main chain hydrogen bonding (see above), are seen in all subunits (e.g., Fig. 8) and in part represent differences between the two protein complexes in side chain hydrogen bonding in this region of the subunit interface. Specifically, the intersubunit hydrogen bond between Glu81 and Ser25 side chains (see above) is present in only two of the four subunits of the two complete des Q58Vdimers—replaced in one chain by a hydrogen bond between a carboxyl oxygen of Asp77 and a ring –NH of His80 on its partner chain (see below).
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-sheet segments, as evidenced by the differences in hydrogen bonding to the terminus of Arg66 (Table 3).
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6.6 ppm not present in the WT protein under similar conditions (Fig. 9). The intensity of the signal and its response to concentration was almost identical to that of the 6.4 ppm dimer signal from Cys28, indicating its association with dimer; its chemical shift was independent of pH in the range 6–8. A similar signal was present in the unliganded dimers of Q58A, G and D mutants (data not shown) and was retained in the unliganded double mutant des 1–6Q58V (Figs. 4, 9), but was absent in the unliganded double mutant Q55A/Q58V (Fig. 10), as well as in the liganded state (Fig. 4) and in mutants (liganded or unliganded) representing single mutations at other positions. The signal was abolished by nitration of the sole protein tyrosine, Tyr49 (Fig. 9), while 2D COSY spectra of the unnitrated protein indicated its connectivity to the Tyr 2,6 ring proton region to which the 3,5 ring protons of Tyr49 connect (Fig. 9). The data assign the 6.6-ppm signal to Tyr49 ring protons originating from a new dimer conformer (see Discussion). Changes near 0.4 ppm also accompanied the Q58 mutations, but were not unambiguously assigned.
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6.65 ppm on or just upfield from the main Tyr49 3,5 proton signal (located at 6.75 ppm). However, at slightly higher pH values, it moved further upfield so that it appeared as a more discrete peak that was similar to, but somewhat less resolved than, the 6.6 ppm signal seen in Q58 mutants (see Discussion). Evidence suggestive of an effect of the des 1–6 mutation on conformation was also seen by CD, the mutation increasing the conformationally sensitive 245/280 nm disulfide band ratio (see Materials and Methods) by 20% (data not shown).
Evidence of yet a different mutation-induced conformational change was seen in the case of the Q55A mutant, the NMR spectra of which showed two new peaks in the unliganded state, one at 0.65 ppm and another at 7.0 ppm (data not shown), the 0.65-ppm peak also seen in the liganded state. Both signals were lost upon nitration of Tyr49, consistent with a tentative assignment of the downfield peak to Tyr49, but the upfield peak remains ambiguous. Most clearly indicative of a conformational effect of the Q55A mutation is that, as noted above, the 6.6 ppm peak of the Q58V mutant is absent in the Q55A/Q58V double mutant (Fig. 10).
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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40% of that value (compare Kanmera and Chaiken 1985; Fassina and Chaiken 1988). Considerable evidence, both from kinetics—which indicate faster binding to dimer than to monomer (Pearlmutter and Dalton 1980)—and from NMR structural studies (see Introduction), argues that differences between unliganded monomeric and dimeric states in NP conformation contribute to the increased binding affinity of the dimer. While such differences could, in principle, be sufficient in themselves to also account for the increased dimerization constant of the liganded state, the present results will be shown to represent evidence that ligand-induced changes involving the interdomain loop—particularly those leading to changes in the hydrogen bonding of residues 58, 56, and 55 and in interactions of the loop (and possibly also residues 53–54) with the amino terminus—potentially contribute to the ligand-induced increase in dimerization. Results will be interpreted in the context of the BNPI crystal structures, which additionally provide new evidence for a role for ligand-induced changes at the subunit interface as part of the allosteric mechanism. Effects of loop mutations not obviously related to allosteric mechanism will be discussed solely to the extent that they provide new insights into neurophysin properties.
Crystal structures of liganded desBNPI and its Q58V mutant and of unliganded desBNPIF91STOP: Ligand-induced changes at the subunit interface
Because interpretation of much of the data obtained rests on the crystal structures of liganded and unliganded BNPI, we point out here that significant differences are often seen among the individual chains of the crystals’ multichain asymmetric units, which we discuss as relevant. These differences are likely to reflect both crystal packing forces and the heterogeneity of monomer and dimer conformers in solution, the latter demonstrated by NMR in both unliganded monomeric and dimeric states (Breslow et al. 1992; Nguyen and Breslow 2002). Note that available data on the bovine neurophysins (for example, see Nicolas et al. 1980) argue against the presence in solution of any unliganded states higher than dimer under the conditions used here and that we have recently confirmed this by analytical ultracentrifugation for the strongly dimerizing des 1–6 Q58V mutant. This is also true for studies of the liganded state by ultracentrifugation (for example, see Nicolas et al. 1980), and fluorescence anisotropy studies have suggested that only trace quantities of liganded oligomers higher than dimer are present at 0.1 mM (Breslow et al. 1991), the upper concentration limit for most binding studies here (Table 2). Also, because we assume the lack of such higher oligomers in solution, the structures of E subunits, which are likely present only in such ensembles, are omitted from the present analyses, as also noted above.
The elucidation of the crystal structure of desBNPIfy provides the first structure of this protein in the liganded state, where, in regions important to its physiological role, it strongly resembles structures of liganded BNPII, with which it shares extensive sequence and functional similarity (see above). The most notable difference between the two structures so far in fact reflects an underlying similarity. That is, despite the differences between the two proteins in interface residues 77 and 81 (Fig. 1), an additional hydrogen bond involving the side chain of residue 81 forms across the interface in the liganded state of both proteins, albeit with different hydrogen bond partners. The binding-induced formation of a new interface hydrogen bond from Thr81 in BNPII was previously noted as a potential contributor to the increased dimerization constant of the liganded state, but, given that the same bond was not possible in BNPI, its importance was uncertain (Wu et al. 2001). The formation of a different ligand-induced interface hydrogen bond by residue 81 in BNPI increases the likelihood that this mechanism contributes significantly to the increased dimerization constant of the liganded state in both neurophysins.
In further support of the significance of this hydrogen bond, the finding that it is present in only three quarters of the chains of the two desBNPIfy dimers is paralleled by a similar situation in the dipeptide complex of BNPII, the asymmetric unit of which also contains two dimers (see above). Even in the Q58V mutant of desBNPI—which required different crystallization conditions from the WT protein, and which contains only two of the three ligand-induced hydrogen bonds between Ser25 and Glu81 found in the WT protein—a different ligand-induced interface hydrogen bond is present in a third subunit, so that the total number of ligand-induced new interface hydrogen bonds is the same. Nonetheless, the universality of this mechanism in other neurophysins remains to be demonstrated, since several do not contain polar residues in position 81 (for example, see Chauvet et al. 1983). Moreover, although formation of this hydrogen bond is the largest ligand-induced interface change, it is not the only one. Multiple small interface adjustments of unmeasured significance accompany ligand binding, as evidenced by RMSD comparison of liganded and unliganded BNPI (see above) and as also reported for BNPII (Wu et al. 2001).
Effects of mutation of Lys59: Role of residue 59 in folding
Lys59 joins the interdomain loop to the carboxyl domain, its carbonyl oxygen involved in the first -sheet hydrogen bond of that domain. The present studies indicate a significant role for Lys59 in the folding pathway and represent the first mutation-induced folding pathway defect seen in NP. The fact that the K59G mutant, but not the K59A or other mutants, required a longer equilibration time with thiol than WT protein to fold normally (see Results) strongly suggests a role for position 59 in guiding the folding path of the carboxyl domain—the smaller range of phi, psi angles preferentially experienced by Ala or Lys residues than by Gly residues most likely reducing the frequency or stability of "incorrect" disulfide partners during folding, decreasing the time needed for disulfide equilibration to the correct structure.
Mutation of Gly57: Effects of steric hindrance and phi, psi angles on binding and dimerization
G57S and G57R mutations of human vasopressin-related NP are a cause of familial neurogenic diabetes insipidus (Ito et al. 1991; Rittig et al. 1996). Effects on peptide binding of mutating residue 57 to Ser or Arg in BNPI (Table 1) have previously been discussed in the context of BNPII crystal structures and assigned to steric hindrance by the mutated residues in the liganded state (Eubanks et al. 2001). The BNPI crystal structures support this explanation of the binding effects but, as with BNPII structures, predict only minor steric hindrance effects of the G57S mutation on the unliganded dimer. The similar effects on dimerization of the Ser and Arg substitutions (Table 1) also argue against a strictly steric hindrance effect. These considerations and the sensitivity of dimerization to loop conformation as demonstrated by the effects of other loop mutations (see below) suggest that changes in loop phi, psi angles or flexibility, arising solely from the substitution of Gly57 by amino acids with more restricted conformations, might account for the effects on dimerization of mutation at this position.
Mutation of Ser56: Effects of side chain hydrogen bonding
Residue 56 mutants were chosen to explore a potential functional role of Ser56 side chain hydrogen bonding as well as the earlier observation that succinylation of Ser56 removed the concentration dependence of binding, implying a loss of ligand-facilitated dimerization (Huang et al. 1993). The 50% decrease in binding constant accompanying mutation of Ser56 to either Gly or Ala (Table 1) is consistent with the fact that, in the liganded state, the –OH of Ser56 is inaccessible to solvent and is hydrogen bonded in all subunits of both BNPI and BNPII to the carbonyl oxygen of Cys21 (Table 4). In contrast, Ser56 is solvent accessible in the unliganded dimer and hydrogen bonds between Ser56 and Cys21 are absent, replaced in 50% of subunits by hydrogen bonding of the Ser –OH group to Gln58 (Table 4). The absence of the –OH in the Gly and Ala mutants therefore leaves the Cys21 oxygen without a hydrogen bond in the nonpolar environment of the liganded state, but has a less destabilizing impact on the unliganded state because of the smaller number and greater solvent accessibility of Ser –OH hydrogen bonds to other residues. In fact, the unliganded dimer gains stability relative to monomer by deletion of the Ser56 –OH group. The dimerization constant of the S56A mutant is increased relative to WT in the unliganded state, suggesting that the few Ser56 –OH hydrogen bonds in the unliganded WT dimer destabilize the unliganded dimer relative to monomer. This is confirmed and its significance discussed below in the context of the role of Gln58.
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50%, an effect similar to that of succinylation (Eubanks et al. 2001), but decreased binding affinity to an extent even greater than the 95% produced by succinylation, precluding measurement of the extent of allostery in the mutant. The effect on binding is possibly the consequence of a deeper burial of the negatively charged Glu carboxyl than of the hydroxyl-esterified succinyl group in the nonpolar environment of the bound state, but a 30% lower than normal 245/280 nm disulfide CD ratio for this mutant (see Materials and Methods) suggests an altered conformation as well.
Effects of mutating Gln58: Evidence for a role in allosteric mechanism
Comparison of BNPI and BNPII crystal structures indicates that the environment of Gln58 similarly identifies the state of dimer ligation in both proteins. (Investigation of the liganded monomeric state is so far precluded by its high dimerization constant.) In the unliganded dimeric states of both proteins, the Gln58 carboxamide faces the protein interior, in relatively close proximity (5 Å) to the ring edge of Phe22, as shown specifically for BNPI in Figure 11. This is even more the case for the H80E mutant in its unliganded monomeric state (Nguyen and Breslow 2002; H. Lee, M.T. Naik, C. Bracken, and E. Breslow, in prep.), where the Gln58 terminus is only 3.5 Å from the Phe22 ring edge. However, in unliganded dimeric BNPI and BNPII structures, but not thus far seen in the unliganded H80E monomer, the Gln58 carboxamide nitrogen is hydrogen bonded to the backbone oxygens of Ser56 and Gly57, while its carboxamide oxygen hydrogen bonds in one des BNPI subunit to the Ser56 hydroxyl (Fig. 11; Table 4). In contrast, the Gln58 side chain projects into the solvent in the liganded dimeric state of both proteins, its carboxamide now 8–9 Å from the Phe22 ring and not hydrogen bonded to another residue (Fig. 11; Table 4).
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To explain the effect of these mutations on dimerization, we note that all of the Q58 mutations have a similar effect on the conformation of the unliganded dimer (see Results). Moreover, what they also have in common is that they either lack side chains capable of hydrogen bonding (Gly, Ala, Val mutants) or (in the case of the Asp mutant) of forming the same hydrogen bonds that the Gln58 terminus forms in the WT unliganded dimer. These findings suggest that the Gln58 hydrogen bonds in the unliganded dimer constrain its conformation and reduce its stability advantage over monomer, loss of these hydrogen bonds on average doubling the dimerization constant of the Gly, Ala, and Val mutants and changing the conformation of the unbound dimer. This interpretation is strongly supported by the increased dimerization constant of the S56A mutant (see above), which has similarly lost the hydrogen bond between the Ser hydroxyl and Gln58 terminus and additionally has lost a hydrogen bond between the Ser56 hydroxyl and the backbone N of Gln58 that might also destabilize the dimer (Table 4). (The fact that the dimerization constants of the S56G and Q58D mutants are not obviously increased relative to WT [Table 1] suggests negative effects on dimerization of the increased flexibility associated with the S56G mutation and of electrostatic repulsion arising from the internalized carboxyl of the Q58D mutant.) Most importantly, since the Gln58 hydrogen bonds are also lost when Gln58 is externalized by ligand binding, the results suggest that the ligand-induced conformational change in Gln58 contributes a factor of approximately two to the ligand-induced 100-fold increase in dimerization constant.
The origins of the high binding constant of the Q58V mutant are not definitively established by the present studies, but the data suggest that the explanation lies principally in the properties of the liganded state, as opposed to a mutation-induced destabilization of the unliganded state. The binding difference cannot be explained solely by the NMR difference between the WT protein and the Q58V mutant in their conformations in the unbound state, since this difference and the accompanying higher dimerization constant is shared by other Q58 mutants (Q58A and Q58G) that do not have high binding constants. It also seems unlikely that the single difference between the two proteins in dimer interface hydrogen bonding in the bound state accounts for their different binding properties. The Q58V mutation similarly increases the binding affinities of WT and des 1–6 BNPI, which were determined at pH 6.2 and 7.4, respectively (Table 1). However, the His80 ring should be a significantly stronger hydrogen bond donor in the protonated state than in the unprotonated state—all the more so in this case because the hydrogen bond involves the carboxyl oxygen of Asp77—and, at least in the WT protein, has a pK value of 6.87 in the unliganded state (Griffin et al. 1975). A significantly lower effect of the mutation on binding by the des 1–6 protein than on WT would therefore have been expected.
We suggest instead that the stronger binding by the mutant might result directly from the effects of the mutation on loop backbone hydrogen bonding (Table 3), the increased number of hydrogen bonds in the mutant—if not compensated for by differences elsewhere in the protein—having the potential to directly increase the stability of the liganded mutant relative to that of the liganded WT protein. This suggestion does not in itself explain why the binding constants of the Q58A and G mutants are not similarly elevated. However, preliminary modeling suggests that the answer might lie in a less restricted local loop conformation in the bound state for these mutants (as well as for WT) relative to that in the branched chain Val mutant, providing greater entropy but diminishing the enthalpic stability of adjacent hydrogen bonds.
Effects of deletion of residues 1–6: Potential allosteric role of the amino-terminal tail
Table 1 demonstrates that, as with BNPII (Zheng et al. 1997), removal of the amino-terminal six residues in BNPI increases binding and dimerization, both by a factor of approximately two in the WT protein. Thus, interactions between the amino terminus and the 53–59 sequence reduce the binding and dimerization constants of the unliganded state. Because such interactions are known to be lost in the liganded state of BNPII (see above), where excision of residues 1–6 leads to a fourfold increase in dimerization constant, the results argue that, in the case of BNPII, loss of amino terminus interactions contributes a factor of approximately four to the ligand-induced increase in dimerization constant. A caveat is that, in contrast to the effects of excision, residues 1–6 remain covalently attached to liganded full-length NP. However, in crystal structures of complexes of WT BNPII (see Introduction), residues 1–6 are unresolved in most chains, suggesting that they are disordered. In chains where they are resolved, no interactions with other regions of the protein are seen. A significant effect on dimerization of the covalent attachment of residues 1–6 in the liganded state therefore seems unlikely.
In the case of BNPI, no firm evidence was obtained for loss of interactions involving the amino-terminal tail in the liganded state, in part because the crystal data represent only the des 1–6 protein. However, the crystal data indicate an average ligand-induced increase of 2 Å in the distance between residues 55 and 7, consistent with such a loss. Moreover, the fact that, at least in the monomer structure (see above), these interactions involve binding site residues 53 and 54 in addition to loop residues increases the likelihood of their loss in the liganded state. The lack of more definitive evidence in part reflects the lack of influence of these interactions on 1D NMR spectra of unliganded BNPI (see above), a probable result of the difference in amino-terminal sequences of BNPI and BNPII (Fig. 1) and the consequences of this for the nature and spectral effects of the interaction. The data therefore allow, but do not confirm, a contribution of a factor of approximately two from loss of amino terminus interactions to the ligand-induced increase in BNPI dimerization.
Long range nature of the effects of Gln58 mutation and of deleting residues 1–6
Both Gln58 mutations and deletion of residues 1–6 lead to changes in Tyr49 NMR spectra in the unliganded dimeric state. Although the proximity of Tyr49 to the 1–6 sequence is unknown in this state, the distance of closest approach between Tyr49 and Gln58 is 20 Å. The chemical shifts of the altered Tyr49 signals and the pH dependence of that in the des 1–6 derivatives suggest that they represent a subset of previously observed conformers of Tyr49 in unliganded dimeric BNPI, found to differ in WT and des 1–8 proteins and thought to be influenced by the titration of His80 (Breslow et al. 1992), the latter 15 Å from Tyr49. In all, these data indicate that effects on the unliganded dimeric state of Gln58 mutation and of excision of the amino terminus represent long-range effects on protein conformation. They additionally suggest that effects on Tyr49 NMR signals upon excision of residues 1–8, originally attributed to the loss of Arg8 (Peyton et al. 1986; Breslow et al. 1992), arise at least in part from the loss of residues 1–6.
The mechanism by which mutation or other change in status of residue 58 affects dimerization is likely to involve Phe22 (e.g., Fig. 11), which contacts residues in both the amino and carboxyl domains that form paths of noncovalent linkages to the subunit interface. This suggestion is supported by the previously demonstrated dimerization-induced change in the contact of Phe22 to carboxyl domain residue Ala68 (for example, see Nguyen and Breslow 2002), which has van der Waals contacts to interface residues in both monomeric and dimeric states. Consistent also with such a role for Phe22, the distance between Gln58 and Phe 22 differs in each of the three relevant protein states—unbound monomer, unbound dimer, and bound dimer (see above). On the other hand, a specific mechanism by which both Gln58 mutation and excision of residues 1–6 might affect Tyr 49 conformers—other than altering conformational constraints at the end of the 3, 10 helix of which Tyr49 is the terminus—is less apparent.
Effects of Gln55 mutation: Evidence of differences between BNPI and BNPII
Interactions of the residue 55 side chain are nonidentical in the different bovine NP complexes, some of these differences reflecting the presence or absence of a third residue in the ligand and its identity if present (Wu et al. 2001). However, these interactions differ most significantly on comparison of the unliganded states of the two bovine neurophysins (Table 4). In the crystal structure of unliganded des 1–6 BNPII, no side chain atoms of Gln55 are visible beyond the beta position, suggesting that they are disordered. In crystals of unliganded desBNPIF91STOP, the carboxamide terminus of Gln55 is completely identified and shown to be hydrogen bonded in at least half of dimer chains (Table 4). These differences suggest that residue 55 either plays no role in allosteric mechanism or that its role is different in different neurophysins.
The Q55A mutation is of particular interest in this context. The increase in dimerization constant resulting from mutation to Ala indicates that the 
-CH2 and/or the carboxamide of Gln55 negatively affect dimerization. The NMR structure of the H80E monomer indicates no hydrogen bonding of the Gln55 side chain. The data accordingly indicate that, as with Gln58, the weaker dimerization of the WT protein than of the Q55A mutant reflects constraints on dimerization placed by the specific bonding interactions of the Gln terminus in the unliganded dimer. Also, analogous to the effects of Gln58 mutations, release of these constraints by mutation appears manifest by conformational change in the unliganded state (see Results). Therefore, since these particular hydrogen bonding interactions are lost in the liganded state (Table 4), the results tend also to suggest that this loss contributes to the ligand-induced increase in BNPI dimerization constant and that the contributions of Gln55 to ligand-facilitated dimerization differ in BNPI and BNPII.
There are two caveats, however. One is that, in contrast to the Gln58 side chain, the Gln55 carboxamide has significant bound-state interactions in both BNPI and BNPII (Table 4), which might have an effect of their own on dimerization in both proteins. The importance of these contacts to the stability of the bound state is one probable cause of the reduced binding constant of the Q55A mutant (Table 1). To the extent that they might also alter dimerization, their impact on the net contribution of ligand-induced changes in Gln55 to the ligand-induced increase in dimerization needs to be considered for both BNPI and BNPII. Second, although Gln55 side chain hydrogen bonds in the unliganded state serve to constrain dimerization only in BNPI, their quantitative contributions even in BNPI are ambiguous given the extreme nonadditivity of Gln55 and Gln58 mutation effects on dimerization (Table 2)—an effect likely to reflect the nonadditivity of their effects on conformation in the unliganded state (see Results) or (less likely given their different NMR effects) the possibility that the two mutations act via a common process. Accordingly, any quantitative difference between the two neurophysins in the contribution of Gln55 to ligand-facilitated dimerization remains to be demonstrated.
With respect to nonadditivity, it is also relevant to point to the nonadditivity of effects of the Q55A and des1–6 mutations, particularly but not exclusively on binding (Table 2). Given the NMR-demonstrated contacts between residue 55 and the 1–6 region (see Introduction), this nonadditivity is not surprising and supports the view that the effects of the 1–6 region on dimerization are mediated at least in part via its interactions with residue 55. A potential explanation of the nonadditivity would invoke a tightening of these interactions in the Q55A mutant. Since these interactions impede both binding and dimerization, this effect would contribute to the low binding constant of the Q55A mutant and reduce the dimerization constant of the Q55A mutant relative to what it would be in its absence. Loss of this effect in the double mutant would increase both its binding and dimerization constants relative to those predicted by additivity, as observed.
Significance and conclusions: Importance of loop interactions to allosteric mechanism
The 100-fold increase in NP dimerization upon occupancy of the hormone-binding site represents an increase in the standard negative free energy of NP dimerization of 2.8 kcal/mol, a value that includes the as yet unquantitated contribution of dimerization-induced changes in the unliganded state that increase binding affinity. The present studies reveal two contributions to the 2.8 kcal/mol additional to the effects of dimerization on binding affinity—a ligand-induced change in the nature of the interface and the presence of loop and amino terminus interactions that constrain dimerization in the unliganded state and that are absent in the liganded state. First, the new crystal structures demonstrate the ligand-induced formation of an additional intersubunit hydrogen bond by residue 81. Together with earlier studies of des 1–6 BNPII (Wu et al. 2001), these results provide the clearest evidence to date of a ligand-induced change in the interface of potential energetic significance and an explanation of earlier analysis of pressure-induced NP dimer dissociation, which suggested an extension of the subunit interface or a decrease in its water accessibility upon ligand binding (Breslow et al. 1991). Second, the data demonstrate that, in BNPI, the side chain hydrogen bonding of loop residues 55, 56, and 58 and the interactions of the amino terminus with the loop (particularly with residue 55), and possibly also with residues 53 and 54, reduce the dimerization constant of the unliganded state and that most, but not necessarily all, of these interactions are lost or altered in the liganded state. Similarities and potential differences between BNPI and BNPII in the details of loop involvement are observed.
The separate contribution of ligand-induced changes of the subunit interface to ligand-facilitated dimerization cannot yet be calculated, in part because its independence from loop contributions is unknown; e.g., effects on dimerization of changes in the loop might include interface changes. However, the potential role of ligand-induced changes in loop and amino terminus interactions, regardless of other effects they might generate, is significant. In BNPI, the combined constraints on dimerization of the unliganded state generated by hydrogen bonding interactions of Gln55, Gln58, and Ser56 side chains and by interactions of the amino terminus are 1 kcal/mol as judged by the increased dimerization associated with the double mutant Q55A/Q58V and the des 1–6 mutation (e.g., Table 2). In BNPII, where the Gln55 side chain does not appear to be involved in the unliganded state, but where the consequences for dimerization of excision of the amino terminus are twice as high as in BNPI (see above), estimates of loop constraints on dimerization in the unliganded state range from 0.8 to 1.3 kcal/mol depending on assumptions made about Gln58; hydrogen-bonding interactions of the Gln58 terminus in the two proteins are similar but not identical (Table 3).
The realized contribution of these constraints to the 2.8 kcal/mol ligand-induced increase in the negative free energy of dimerization depends on the extent to which these constraints are actually lost in the liganded state. In BNPII, in which ligand-induced release of both amino terminus interactions and Gln58 carboxamide hydrogen bonding is demonstrable, these contributions should approximately equal loss of the constraints they impose on dimerization in the unliganded state—roughly 40% of the total change in dimerization free energy. In BNPI, these contributions range from 19% to 36% of the total free energy change depending on whether amino terminus interactions are indeed lost in the liganded state. Potential bound state contributions of the Gln55 terminus in both proteins remain to be evaluated.
The present results also have implications for the mechanism of dimerization in the unliganded state, specifically providing evidence that such dimerization involves formation of intraloop hydrogen bonds that constrain both the dimerization constant and conformation of the unliganded state. The inability to form these hydrogen bonds increases dimerization, albeit to a conformationally altered state. The results underscore the importance of loop interactions to neurophysin conformation in general and to dimerization in particular and are consistent with their potential role in domain orientation (see Introduction). A specific path involving Phe22 is suggested by which the status of the loop at residue 58 is communicated to the rest of the protein.
The mechanism by which specific interactions constrain neurophysin dimerization in the unliganded state contrasts with that operative in epidermal growth factor receptor (for example, see Dawson et al. 2005). In the receptor case, the unliganded state is monomeric, and such interactions directly restrain residues that ultimately form part of the dimer subunit interface in the liganded state. With neurophysin, the constraints operate at a distance on the subunit interface to maintain a low but significant dimerization constant in the unliganded state. Release of these constraints upon ligand-binding is mediated by proximity of the constraining interactions to the binding site and leads principally to optimization and enhancement of preexisting interchain contacts.
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Materials and methods |
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The WT and mutant proteins were expressed in E. coli and isolated from inclusion bodies in their misfolded state also as described earlier (Eubanks et al. 1999, 2000). The misfolded state is a disulfide-scrambled state and is normally folded at pH 8 by stirring in air for 48 h, in the presence of -mercaptoethanol to facilitate disulfide exchange and ligand peptide to drive the folding reaction (Eubanks et al. 2000). This procedure was used successfully for all mutants, but gave low folding yields (
50% of normal) for the K59G and S56E mutants. The low yield of the S56E mutant was shown to reflect a low affinity for peptide (see Results). However, yields for the K59G mutant were restored to normal by folding in a glutathione buffer (3 mM GSSG, 2 mM GSH, 10 mM FY in 0.1 M sodium acetate at pH 7.4) in the absence of air (see Discussion). After folding, protein was separated from the other components of the folding mixture by gel filtration and lyophilized. With the exception of the S56E mutant, correctly folded protein was separated from misfolded protein by chromatography on a ligand-linked affinity column and dialyzed and lyophilized as previously described (Eubanks et al. 2000). Affinity chromatography could be not used for the S56E mutant because of the low peptide affinity of its folded state, and the folded state of this protein was separated from misfolded states by HPLC as previously described for other mutants that retain some folding ability but that cannot bind peptide (Eubanks et al. 2001). Masses of all purified folded proteins were confirmed by mass spectrometry. Folding was confirmed by circular dichroism; all folded mutants exhibited the normal two conformationally sensitive disulfide signals at 245 and 280 nm with—except as indicated for the S56E and des1–6 mutants (see Results and Discussion)—chain molar ellipticities of +20,000 and –20,000 deg cm2/dmol, respectively (for example, see Eubanks et al. 2001).
Determination of dimerization constants
Dimerization constants were measured by NMR at 25°C as previously described (for example, see Zheng et al. 1997; Eubanks et al. 2000), using the relative intensities of Cys28 
-proton signals, located at 6.15 and 6.4 ppm in monomer and dimer, respectively. Data were obtained on a Varian Inova 600 MHz spectrometer. For most runs, samples were dissolved in D2O containing 10 mM pH 6.2 phosphate buffer readjusted with NaOD or DCl to a final pH of 6.2 (uncorrected electrode reading in D2O) after addition of protein. Several reported studies were carried out without buffer, but at measured pH. Protein concentrations were determined from the CD intensity at 280 nm, assuming a molar (chain) ellipicity of –20,000 deg cm2/dmol (see above). Comparison of concentrations so calculated with values obtained from 280 nm absorbance measurements indicated that this method gave reliable values even for proteins in which the 245/280 ratios were atypical, and was preferred because of its insensitivity to random contaminants from solvent. To obtain the weight ratios (M/D) of monomer (M) to dimer (D), spectra were processed by different methods to reduce effects of noise and baseline uncertainties and then magnified. Intensities of monomer and dimer signals in the magnified spectra were determined by weight. Dimerization constants calculated for individual experiments are averages of the different processing methods. Spectra were obtained at different concentrations as needed to reduce ambiguities.
Other NMR methods
DQF-COSY experiments (Rance et al. 1983) of Q58V and nitrated Q58V consisted of 256 t 1 increments. Spectral widths in both dimensions were 6999.7 Hz. The concentrations of Q58V and nitrated Q58V were 1.5 mM and 0.7 mM, respectively. Processing of the NMR data was carried out by NMRpipe (Delaglio et al. 1995) and analyzed by the Sparky program (SPARKY 3, T.D. Goddard and D.G. Kneller, University of California, San Francisco).
Determination of binding affinities
Peptide binding was measured by CD as previously described (Breslow et al. 1973) using 0.1 mM mononitrated protein that had been prepared as also previously described and further purified by affinity chromatography (Rabbani et al.1982). For most proteins, measurements were made at pH 6.2 in 0.1 M ammonium acetate containing 2 mM MES buffer. However, the peptide complexes of the des 1–6 mutants became insoluble under these conditions, so studies of these proteins were conducted at pH 7.4 in 50 mM Tris acetate, 50 mM ammonium acetate. In all cases, results with the mutant are compared with those of the WT protein under identical conditions.
Binding constant measurement involves determination from the nitrotyrosine CD signal of the fraction of protein in the liganded state at different concentrations of total peptide. Protein concentrations are determined by nitrotyrosine absorbance. Free peptide concentrations are calculated as the difference between bound peptide and total peptide. Because of the linkage between binding and dimerization, Scatchard plots of binding data exhibit a slight curvature that becomes more marked at lower degrees of protein saturation. Also, estimates of free peptide concentration become less reliable at low degrees of protein saturation for strongly binding proteins. Accordingly, for comparison of different peptides or different proteins, as is the case here, we have typically not utilized data obtained at levels of fractional protein saturation (
) <0.4 in binding constant calculations. Constants are instead calculated from Scatchard plot slopes at and above values of = 0.5 (for example, see Breslow et
