Intramembrane-cleaving proteases (I-CLiPs) play crucial roles in physiological and pathological processes, such as Alzheimer’s disease and cancer. However, the mechanisms of substrate recognition by I-CLiPs remain poorly understood. The aspartic I-CLiP presenilin is the catalytic subunit of the γ-secretase complex, which releases the amyloid-β peptides (Aβs) through intramembrane proteolysis of the transmembrane domain of the amyloid precursor protein (APPTM). Here we used solution NMR to probe substrate docking of APPTM to the presenilin homologs (PSHs) MCMJR1 and MAMRE50, which cleaved APPTM in the NMR tube. Chemical shift perturbation (CSP) showed juxtamembrane regions of APPTM mediate its docking to MCMJR1. Binding of the substrate to I-CLiP decreased the magnitude of amide proton chemical shifts δHat the C-terminal half of the substrate APPTM, indicating that the docking to the enzyme weakens helical hydrogen bonds and unwinds the substrate transmembrane helix around the initial ε-cleavage site. The APPTM V44M substitution linked to familial AD caused more CSP and helical unwinding around the ε-cleavage site. MAMRE50, which cleaved APPTM at a higher rate, also caused more CSP and helical unwinding in APPTM than MCMJR1. Our data suggest that docking of the substrate transmembrane helix and helical unwinding is coupled in intramembrane proteolysis and FAD mutation modifies enzyme/substrate interaction, providing novel insights into the mechanisms of I-CLiPs and AD drug discovery.
Serine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.
more » « less- NSF-PAR ID:
- 10360587
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
more » « less
Abstract While immunoglobulins find ubiquitous use in biotechnology as static binders, recent developments have created proantibodies that enable orthogonal switch‐like behavior to antibody function. Previously, peptides with low binding affinity have been genetically fused to antibodies, to proteolytically control binding function by blocking the antigen‐binding site. However, development of these artificial blockers requires panning for peptide sequences that reversibly affect antigen affinity for each antibody. Instead, a more general strategy to achieve dynamic control over antibody affinity may be feasible using protein M (ProtM) from
Mycoplasma genitalium , a newly identified polyspecific immunity evasion protein that is capable of blocking antigen binding for a wide range of antibodies. Using C‐terminus truncation to identify ProtM variants that are still capable of binding to antibodies without the ability to block antigens, we developed a novel and universal biological switch for antibodies. Using a site‐specifically placed thrombin cut site, antibody affinity can be modulated by cleavage of the two distinct antibody‐binding and antigen‐blocking domains of ProtM. Because of the high affinity of ProtM toward a large variety of IgG subtypes, this strategy may be used as a universal approach to create proantibodies that are conditionally activated by disease‐specific proteases such as matrix metalloproteinases. -
more » « less
Mechanistic understanding of DNA recombination in the Cre
-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTDand CreCat) to be structurally independent. Amide15N relaxation measurements of the CreCatdomain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity viatrans protein–protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packsin cis over the protein DNA binding surface and active site. Moreover, binding toloxP DNA induces a conformational change that dislodges the C terminus, resulting in acis -to-trans switch that is likely to enable protein–protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency. -
Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L–11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a β site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the β site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).more » « less
-
NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C β and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.more » « less
