Anmin Tan

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.

Evidence for a Ca2+-Specific Conformational Change in Avian Thymic Hormone, a High-Affinity β-Parvalbumin

Named for the capacity to stimulate differentiation and maturation of T-cell precursors, avian thymic hormone (ATH) is nonetheless a beta-parvalbumin that is also expressed in the avian retina. With Ca(2+)- and Mg(2+)-binding constants in excess of 10(8) and 10(4) M(-1), respectively, both EF-hand motifs qualify as Ca(2+)/Mg(2+) sites. However, whereas addition of either apo- or Mg(2+)-bound ATH to 1,8-anilinonaphthalenesulfonic acid (ANS) causes a large increase in quantum yield and a pronounced blue shift, addition of the Ca(2+)-bound protein is without effect. These observations suggest that apo- and Mg(2+)-bound ATH adopt conformations distinct from the Ca(2+)-bound protein, exposing apolar surface for interaction with ANS. Differential scanning calorimetry (DSC) data imply that unfolding of apo-ATH is accompanied by diminished exposure of apolar surface, relative to Ca(2+)-free rat beta-PV, perhaps due to greater solvent-accessible apolar surface in the native form. The fluorescence and DSC results, considered together, may indicate that the AB and CD-EF domains of ATH are not tightly associated in the absence of bound Ca(2+). Consistent with this idea, sedimentation velocity data reveal that the apo- and Mg(2+)-bound forms of ATH show greater departures from spherical symmetry than the Ca(2+)-bound state. These findings suggest that a high-affinity binding signature does not require that the parvalbumin apo- and Ca(2+)-bound conformations be indistinguishable, as we have recently proposed. They also suggest that it is possible to engineer a Ca(2+)-dependent conformational change into a high-affinity EF-hand protein, furnishing a mechanism by which the protein could play a reverse Ca(2+) sensor role.

Solution structures of polcalcin Phl p 7 in three ligation states: Apo‐, hemi‐Mg2+‐bound, and fully Ca2+‐bound

Polcalcins are small EF‐hand proteins believed to assist in regulating pollen‐tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain‐swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg2+, or 100 μM Ca2+. Phl p 7 is monomeric in all three ligation states. In the apo‐form, both EF‐hand motifs reside in the closed conformation, with roughly antiparallel N‐ and C‐terminal helical segments. In 5.0 mM Mg2+, the divalent ion is bound by EF‐hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca2+‐bound Phl p 7 resembles that previously reported for Bet v 4—likewise exposing apolar surface to the solvent. Occluded in the apo‐ and Mg2+‐bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF‐hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at –x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca2+. When Mg2+ resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer‐sphere ligand. 15N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states. Proteins 2013.

Divalent ion binding properties of the timothy grass allergen, Phl p 7.

The timothy grass allergen, Phl p 7, was studied by calorimetry, spectroscopy, and analytical ultracentrifugation. As judged by isothermal titration calorimetry (ITC), the protein binds Ca (2+) cooperatively with stepwise macroscopic association constants of 1.73 x 10 (6) and 8.06 x 10 (6) M (-1). By contrast, Mg (2+) binding is sequential with apparent macroscopic association constants of 2.78 x 10 (4) and 170 M (-1). Circular dichroism and ANS fluorescence data suggest that Ca (2+) binding provokes a major conformational change that does not occur upon Mg (2+) binding. Conformational stability was assessed by differential scanning calorimetry (DSC). In phosphate-buffered saline (PBS) containing EDTA, the apoprotein undergoes two-state denaturation with a T m of 78.4 degrees C. In the presence of 0.02 mM Ca (2+), the T m exceeds 120 degrees C. Phl p 7 is known to crystallize as a domain-swapped dimer at low pH. However, analytical ultracentrifugation data indicate that the protein is monomeric in neutral solution at concentrations exceeding 1.0 mM, in both the apo and Ca (2+)-bound states.

Polcalcin divalent ion-binding behavior and thermal stability: comparison of Bet v 4, Bra n 1, and Bra n 2 to Phl p 7.

Polcalcins are pollen-specific proteins containing two EF-hands. Atypically, the C-terminal EF-hand binding loop in Phl p 7 (from timothy grass) harbors five, rather than four, anionic side chains, due to replacement of the consensus serine at -x by aspartate. This arrangement has been shown to heighten parvalbumin Ca(2+) affinity. To determine whether Phl p 7 likewise exhibits anomalous divalent ion affinity, we have also characterized Bra n 1 and Bra n 2 (both from rapeseed) and Bet v 4 (from birch tree). Relative to Phl p 7, they exhibit N-terminal extensions of one, five, and seven residues, respectively. Interestingly, the divalent ion affinity of Phl p 7 is unexceptional. For example, at -17.84 +/- 0.13 kcal mol(-1), the overall standard free energy for Ca(2+) binding falls within the range observed for the other three proteins (-17.30 +/- 0.10 to -18.15 +/- 0.10 kcal mol(-1)). In further contrast to parvalbumin, replacement of the -x aspartate, via the D55S mutation, actually increases the overall Ca(2+) affinity of Phl p 7, to -18.17 +/- 0.13 kcal mol(-1). Ca(2+)-free Phl p 7 exhibits uncharacteristic thermal stability. Whereas wild-type Phl p 7 and the D55S variant denature at 77.3 and 78.0 degrees C, respectively, the other three polcalcins unfold between 56.1 and 57.9 degrees C. This stability reflects a low denaturational heat capacity increment. Phl p 7 and Phl p 7 D55S exhibit DeltaC(p) values of 0.34 and 0.32 kcal mol(-1) K(-1), respectively. The corresponding values for the other three polcalcins range from 0.66 to 0.95 kcal mol(-1) K(-1).

Energetics of OCP1-OCP2 complex formation

OCP1 and OCP2, the most abundant proteins in the cochlea, are putative subunits of an SCF E3 ubiquitin ligase. Previous work has demonstrated that they form a heterodimeric complex. The thermodynamic details of that interaction are herein examined by isothermal titration calorimetry. At 25 degrees C, addition of OCP1 to OCP2 yields an apparent association constant of 4.0 x 10(7) M(-1). Enthalpically-driven (DeltaH=-35.9 kcal/mol) and entropically unfavorable (-TDeltaS=25.5 kcal/mol), the reaction is evidently unaccompanied by protonation/deprotonation events. DeltaH is strongly dependent on temperature, with DeltaC(p)=-1.31 kcal mol(-1) K(-1). Addition of OCP2 to OCP1 produces a slightly less favorable DeltaH, presumably due to the requirement for dissociation of the OCP2 homodimer prior to OCP1 binding. The thermodynamic signature for OCP1/OCP2 complex formation is inconsistent with a rigid-body association and suggests that the reaction is accompanied by a substantial degree of folding.

Solution structures of chicken parvalbumin 3 in the Ca2+‐free and Ca2+‐bound states

Birds express two β‐parvalbumin isoforms, parvalbumin 3 and avian thymic hormone (ATH). Parvalbumin 3 from chicken (CPV3) is identical to rat β‐parvalbumin (β‐PV) at 75 of 108 residues. CPV3 displays intermediate Ca2+ affinity—higher than that of rat β‐parvalbumin, but lower than that of ATH. As in rat β‐PV, the attenuation of affinity is associated primarily with the CD site (residues 41–70), rather than the EF site (residues 80–108). Structural data for rat α‐ and β‐parvalbumins suggest that divalent ion affinity is correlated with the similarity of the unliganded and Ca2+‐bound conformations. We herein present a comparison of the solution structures of Ca2+‐free and Ca2+‐bound CPV3. Although the structures are generally similar, the conformations of residues 47 to 50 differ markedly in the two protein forms. These residues are located in the C helix, proximal to the CD binding loop. In response to Ca2+ removal, F47 experiences much greater solvent accessibility. The side‐chain of R48 assumes a position between the C and D helices, adjacent to R69. Significantly, I49 adopts an interior position in the unliganded protein that allows association with the side‐chain of L50. Concomitantly, the realignment of F66 and F70 facilitates their interaction with I49 and reduces their contact with residues in the N‐terminal AB domain. This reorganization of the hydrophobic core, although less profound, is nevertheless reminiscent of that observed in rat β‐PV. The results lend further support to the idea that Ca2+ affinity correlates with the structural similarity of the apo‐ and bound parvalbumin conformations. Proteins 2011.

Conformational stabilities of guinea pig OCP1 and OCP2.

OCP1 and OCP2, the most abundant proteins in the cochlea, are evidently subunits of an SCF E3 ubiquitin ligase. Although transcribed from a distinct gene, OCP2 is identical to Skp1. OCP1 is equivalent to the F-box protein known as Fbs1, Fbx2, or NFB42 - previously shown to bind N-glycosylated proteins and believed to function in the retrieval and recycling of misfolded proteins. The high concentrations of OCP1 and OCP2 in the cochlea suggest that the OCP1-OCP2 heterodimer may serve an additional function independent of its role in a canonical SCF complex. At 25 degrees C, urea-induced denaturation of OCP1 is slow, but reversible. The data suggest that the protein possesses one or more disordered regions, a conclusion supported by analysis of the far-UV circular dichroism spectrum and the appearance of the (1)H, (15)N-HSQC spectrum. Thermal denaturation of OCP1 is irreversible, evidently due to formation of high molecular weight aggregates. Analysis with a kinetic model yields an estimate for the activation energy for unfolding of 49 kcal/mol. Urea denaturation data for OCP2 returns DeltaG(o) and m values of 6.2 kcal/mol and 1.5 kcal mol(-)(1) M(-1), respectively. In contrast to OCP1, thermal denaturation of OCP2 is reversible. In phosphate-buffered saline, at pH 7.40, the protein exhibits a DeltaH(vH)/DeltaH(cal) ratio of 1.69, suggesting that denaturation proceeds largely from the native dimer directly to the unfolded state. OCP1 and OCP2 associate tightly at room temperature. However, DSC data for the complex suggest that they denature independently, consistent with the highly exothermic enthalpy of complex formation reported previously.

Structure of Avian Thymic Hormone, a High-affinity Avian β-parvalbumin, in the Ca2+-free and Ca2+-bound States

Originally isolated on the basis of its capacity to stimulate T-cell maturation and proliferation, avian thymic hormone (ATH) is nevertheless a parvalbumin, one of two beta-lineage isoforms expressed in birds. We recently learned that addition of Ca(2+)-free ATH to a solution of 8-anilinonaphthalene-1-sulfonate (ANS) markedly increases ANS emission. This behavior, not observed in the presence of Ca(2+), suggests that apolar surface area buried in the Ca(2+)-bound state becomes solvent accessible upon Ca(2+) removal. In order to elucidate the conformational alterations that accompany Ca(2+) binding, we have obtained the solution structure of the Ca(2+)-free protein using NMR spectroscopy and compared it to the Ca(2+)-loaded protein, solved by X-ray crystallography. Although the metal-ion-binding (CD-EF) domains are largely coincident in the superimposed structures, a major difference is observed in the AB domains. The tight association of helix B with the E and F helices in the Ca(2+)-bound state is lost upon removal of Ca(2+), producing a deep hydrophobic cavity. The B helix also undergoes substantial rotation, exposing the side chains of F24, Y26, F29, and F30 to solvent. Presumably, the increase in ANS emission observed in the presence of unliganded ATH reflects the interaction of these hydrophobic residues with the fluorescent probe. The increased solvent exposure of apolar surface area in the Ca(2+)-free protein is consistent with previously collected scanning calorimetry data, which indicated an unusually low change in heat capacity upon thermal denaturation. The Ca(2+)-free structure also provides added insight into the magnitude of ligation-linked conformational alteration compatible with a high-affinity metal-ion-binding signature. The exposure of substantial apolar surface area suggests the intriguing possibility that ATH could function as a reverse Ca(2+) sensor.

Heightened stability of polcalcin Phl p 7 is correlated with strategic placement of apolar residues.

Phl p 7 exhibits atypical conformational stability and a diminutive denaturational heat capacity increment, ΔC(p). Because exposure of apolar surface largely dictates the magnitude of ΔC(p), a depressed value could signify an unusually compact unfolded state. The volume of the denatured state ensemble (DSE) is evidently inversely correlated with mean hydrophobicity [Pace et al., Protein Sci. 19 (2010) 929-943]. Interestingly, apolar residues replace more polar ones at four positions in Phl p 7. We herein examine the consequences of replacing those residues with the corresponding ones from Bra n 1, a related isoform. All four mutations - M4H, L21A, I60T, and C63A - destabilize Phl p 7. Our analysis suggests that the DSE of Phl p 7 is indeed highly compact and that the substitutions act by increasing its volume and solvent-accessibility. All four mutations increase the urea m value; L21A, I60T, and C63A also yield a perceptible increase in ΔC(p).