Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Specific Changes in the Local Flexibility of Plasminogen Activator Inhibitor 1 upon Binding to the Somatomedin B Domain of Vitronectin

Abstract

The native fold of plasminogen activator inhibitor 1 (PAI-1) represents an active metastable conformation that spontaneously converts to an inactive latent form. Binding of the somatomedin B domain (SMB) of the endogenous cofactor vitronectin to PAI-1 delays the transition to the latent state and increases the thermal stability of the protein dramatically. We have used hydrogen/deuterium exchange mass spectrometry to assess the inherent structural flexibility of PAI-1 and to monitor the changes induced by SMB binding. Our data show that the PAI-1 core consisting of β-sheet B is rather protected against exchange with the solvent, while the remainder of the molecule is more dynamic. SMB binding causes a pronounced and widespread stabilization of PAI-1 that is not confined to the binding interface with SMB. We further explored the local structural flexibility in a mutationally stabilized PAI-1 variant (14-1B) as well as the effect of stabilizing antibody Mab-1 on wild-type PAI-1. The three modes of stabilizing PAI-1 (SMB, Mab-1, and the mutations in 14-1B) all cause a delayed latency transition, and this effect was accompanied by unique signatures on the flexibility of PAI-1. Reduced flexibility in the region around helices B, C, and I was seen in all three cases, which suggests an involvement of this region in mediating structural flexibility necessary for the latency transition. These data therefore add considerable depth to our current understanding of the local structural flexibility in PAI-1 and provide novel indications of regions that may affect the functional stability of PAI-1.

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability

“…Peptides comprising the lower backside underlying the shutter (hB and hC), flexible joint region (hD, hE, and s1A), shutter region (s3A and s5A), and gate region (s3C, s4C, and s3B) experience substantial deuterium uptake over the experimental time course (Figure S2). Consistent with previous work, the only regions predominantly protected from exchange exist in β-sheet B in the protein core. Also consistent with previous work, addition of the SMB domain of VN resulted in widespread stabilization of the flexible joint region and the backside, constituting the majority of the lower half of the molecule (Figure S2).…”

“…Binding of PAI-1 by the SMB domain from VN, which houses the primary highaffinity binding site for PAI-1, 20 results in significant protection against exchange in several peptides, namely, 46−59 (hB−hC), 99−113 (s2A−hE), 114−128 (hE−s1A), and 299−306 (hI loop−s5A) (Figures S2 and S3). This is consistent with previously published results, 31 although the SMB domain effect is generally less pronounced in this study because of the reduced dynamics of PAI-1 in MOPS buffer compared to that in PBS. The noted peptides are located in or proximal to the flexible joint region, including the SMB domain-binding interface.…”

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability

“…Peptides comprising the lower backside underlying the shutter (hB and hC), flexible joint region (hD, hE, and s1A), shutter region (s3A and s5A), and gate region (s3C, s4C, and s3B) experience substantial deuterium uptake over the experimental time course (Figure S2). Consistent with previous work, the only regions predominantly protected from exchange exist in β-sheet B in the protein core. Also consistent with previous work, addition of the SMB domain of VN resulted in widespread stabilization of the flexible joint region and the backside, constituting the majority of the lower half of the molecule (Figure S2).…”

“…Binding of PAI-1 by the SMB domain from VN, which houses the primary highaffinity binding site for PAI-1, 20 results in significant protection against exchange in several peptides, namely, 46−59 (hB−hC), 99−113 (s2A−hE), 114−128 (hE−s1A), and 299−306 (hI loop−s5A) (Figures S2 and S3). This is consistent with previously published results, 31 although the SMB domain effect is generally less pronounced in this study because of the reduced dynamics of PAI-1 in MOPS buffer compared to that in PBS. The noted peptides are located in or proximal to the flexible joint region, including the SMB domain-binding interface.…”

Effects of Protein–Ligand Interactions on Hydrogen/Deuterium Exchange Kinetics: Canonical and Noncanonical Scenarios

“…These data reveal that Hb indeed represents a type 2 binder. Our findings are consistent with earlier tritium-labeling and HDX work. , However, those studies did not discuss the unusual nature of Hb–O 2 interactions, which are different from the type 1 binding behavior seen for most other proteins. − ,,, …”

Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry

“…On the contrary, peptide 127–134 (lower helix F) still contains a number of amide hydrogens after 80 minutes, indicating a segment of relative stability in helix F at 5 °C. As previously observed at 25 °C 22 , most regions of PAI-1 are stabilized upon transition to the latent conformation and as a result incorporates less deuterium at 5 °C (Fig. 3, Figures S4 and S5).…”

“…2b). Interestingly, the pattern of flexible and rigid regions obtained through these simulations correlate well with the pattern previously obtained through hydrogen/deuterium-exchange experiments at 25 °C or 37 °C 19, 21, 22 . This is not surprising, as structural flexibility is often associated with solvent exposure and transient breaking of backbone hydrogen bonds, - the two prerequisites for hydrogen/deuterium-exchange on backbone amides.…”

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability

“…Peptides comprising the lower backside underlying the shutter (hB and hC), flexible joint region (hD, hE, and s1A), shutter region (s3A and s5A), and gate region (s3C, s4C, and s3B) experience substantial deuterium uptake over the experimental time course (Figure S2). Consistent with previous work, the only regions predominantly protected from exchange exist in β-sheet B in the protein core. Also consistent with previous work, addition of the SMB domain of VN resulted in widespread stabilization of the flexible joint region and the backside, constituting the majority of the lower half of the molecule (Figure S2).…”

“…Binding of PAI-1 by the SMB domain from VN, which houses the primary highaffinity binding site for PAI-1, 20 results in significant protection against exchange in several peptides, namely, 46−59 (hB−hC), 99−113 (s2A−hE), 114−128 (hE−s1A), and 299−306 (hI loop−s5A) (Figures S2 and S3). This is consistent with previously published results, 31 although the SMB domain effect is generally less pronounced in this study because of the reduced dynamics of PAI-1 in MOPS buffer compared to that in PBS. The noted peptides are located in or proximal to the flexible joint region, including the SMB domain-binding interface.…”

Effects of Protein–Ligand Interactions on Hydrogen/Deuterium Exchange Kinetics: Canonical and Noncanonical Scenarios

“…These data reveal that Hb indeed represents a type 2 binder. Our findings are consistent with earlier tritium-labeling and HDX work. , However, those studies did not discuss the unusual nature of Hb–O 2 interactions, which are different from the type 1 binding behavior seen for most other proteins. − ,,, …”

Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry

“…On the contrary, peptide 127–134 (lower helix F) still contains a number of amide hydrogens after 80 minutes, indicating a segment of relative stability in helix F at 5 °C. As previously observed at 25 °C 22 , most regions of PAI-1 are stabilized upon transition to the latent conformation and as a result incorporates less deuterium at 5 °C (Fig. 3, Figures S4 and S5).…”

“…2b). Interestingly, the pattern of flexible and rigid regions obtained through these simulations correlate well with the pattern previously obtained through hydrogen/deuterium-exchange experiments at 25 °C or 37 °C 19, 21, 22 . This is not surprising, as structural flexibility is often associated with solvent exposure and transient breaking of backbone hydrogen bonds, - the two prerequisites for hydrogen/deuterium-exchange on backbone amides.…”

Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability

“…Peptides comprising the lower backside underlying the shutter (hB and hC), flexible joint region (hD, hE, and s1A), shutter region (s3A and s5A), and gate region (s3C, s4C, and s3B) experience substantial deuterium uptake over the experimental time course (Figure S2). Consistent with previous work, the only regions predominantly protected from exchange exist in β-sheet B in the protein core. Also consistent with previous work, addition of the SMB domain of VN resulted in widespread stabilization of the flexible joint region and the backside, constituting the majority of the lower half of the molecule (Figure S2).…”

“…Binding of PAI-1 by the SMB domain from VN, which houses the primary highaffinity binding site for PAI-1, 20 results in significant protection against exchange in several peptides, namely, 46−59 (hB−hC), 99−113 (s2A−hE), 114−128 (hE−s1A), and 299−306 (hI loop−s5A) (Figures S2 and S3). This is consistent with previously published results, 31 although the SMB domain effect is generally less pronounced in this study because of the reduced dynamics of PAI-1 in MOPS buffer compared to that in PBS. The noted peptides are located in or proximal to the flexible joint region, including the SMB domain-binding interface.…”

Effects of Protein–Ligand Interactions on Hydrogen/Deuterium Exchange Kinetics: Canonical and Noncanonical Scenarios

“…These data reveal that Hb indeed represents a type 2 binder. Our findings are consistent with earlier tritium-labeling and HDX work. , However, those studies did not discuss the unusual nature of Hb–O 2 interactions, which are different from the type 1 binding behavior seen for most other proteins. − ,,, …”

Conformational preludes to the latency transition in PAI-1 as determined by atomistic computer simulations and hydrogen/deuterium-exchange mass spectrometry

“…On the contrary, peptide 127–134 (lower helix F) still contains a number of amide hydrogens after 80 minutes, indicating a segment of relative stability in helix F at 5 °C. As previously observed at 25 °C 22 , most regions of PAI-1 are stabilized upon transition to the latent conformation and as a result incorporates less deuterium at 5 °C (Fig. 3, Figures S4 and S5).…”

“…2b). Interestingly, the pattern of flexible and rigid regions obtained through these simulations correlate well with the pattern previously obtained through hydrogen/deuterium-exchange experiments at 25 °C or 37 °C 19, 21, 22 . This is not surprising, as structural flexibility is often associated with solvent exposure and transient breaking of backbone hydrogen bonds, - the two prerequisites for hydrogen/deuterium-exchange on backbone amides.…”