scholarly journals Cryo-EM reveals disrupted human p97 allosteric activation by disease mutations and inhibitor binding

2021 ◽  
Author(s):  
Brian Caffrey ◽  
Xing Zhu ◽  
Alison Berezuk ◽  
Katharine Tuttle ◽  
Sagar Chittori ◽  
...  

The human AAA ATPase p97, a potential cancer target, plays a vital role in clearing misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders. Here, we present cryo-EM structural analyses of four disease mutants p97R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083. These structures demonstrate that the mutations affect nucleotide-driven p97 allosteric activation by predominantly interfering with either the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), the inter-protomer interactions (p97A232E), or the coupling between D1 and D2 nucleotide domains (p97D592N, p97E470D). We also show that binding of the competitive inhibitor CB-5083 to the D2 domain prevents conformational changes similar to that seen for mutations that affect coupling between D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations.

1981 ◽  
Vol 103 (23) ◽  
pp. 7001-7003 ◽  
Author(s):  
Marjorie E. Winkler ◽  
Konrad Lerch ◽  
Edward I. Solomon

2021 ◽  
pp. 101187
Author(s):  
Brian Caffrey ◽  
Xing Zhu ◽  
Alison Berezuk ◽  
Katharine Tuttle ◽  
Sagar Chittori ◽  
...  

2014 ◽  
Vol 70 (a1) ◽  
pp. C413-C413
Author(s):  
Mahmudul Hasan ◽  
Agnes Rinaldo-Matthis ◽  
Marjolein Thunnissen

Vertebrate leukotriene A4 hydrolases are zinc metalloenzymes with an epoxide hydrolase and aminopeptidase activity belonging to the M1 family of aminopeptidases. Bestatin, an amino peptidase inhibitor, can inhibit both the activities. The human enzyme produces LTB4, a powerful mediator of inflammation and is implicated in a wide variety of rheumatoid diseases. The yeast homolog scLTA4H contains only a rudimentary epoxide hydrolase activity. Both the structure of the human enzyme and recently the structure of scLTA4H and have been solved to investigate the molecular architecture of the active site both with and without inhibitor Bestatin. The structure of scLTA4H shows large domain movements creating an open active site. In the human enzyme the LTA4 binding side is a narrow hydrophobic channel. Upon inhibitor a domain shifts occurs and the final binding pocket is formed. The fact that scLTA4H displays this induced fit is an interesting observation. Many members of the M1 family seem to display a certain degree of induced fit, a feature, which however, has never been observed for humLTA4H. Our recent solution SAXS studies show that humLTA4H does not make any conformational changes upon inhibitor binding which is consistent with our previous speculation that it functions by a lock and key mechanism rather than induced fit and is better suited to supply the protective and precise environment for hydrolysis of LTA4 into LTB4. On the other hand Xenopus LTA4H shows conformational change in the higher/wide angular region ( >1 nm-1) and decrease in Porod volume of approximately 20 nm3 but no change in Rg or Dmax was observed. It is also observed that like in crystal structure Xenopus LTA4H forms dimer in solution. Similarly scLTA4H forms dimer in solution, which is unlike the crystal structure, and also make conformational changes upon inhibitor binding. Taken together, Xenopus and scLTA4H makes more compact form, with decrease in flexibility, to perform it's catalytic action.


1977 ◽  
Vol 55 (8) ◽  
pp. 856-864 ◽  
Author(s):  
T. J. Carne ◽  
T. G. Flynn

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5′-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff s base complex between pyridoxal 5′-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5′-phosphate per mole of PGM dimer through the ε-amino group of a lysyl residue. The effect of pyridoxal 5′-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5′-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5′-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5′-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme.The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.


2021 ◽  
Author(s):  
◽  
Preeti Kundu

<p>Tuberculosis (TB), which is estimated to affect 2 billion individuals worldwide, is an infection predominately caused by Mycobacterium tuberculosis(M. tuberculosis). Of particular concern is the increasing prevalence of TB, which is becoming resistant to the treatments currently available. Anthranilate phosphoribosyltransferase (AnPRT) catalyses the formation of N-(5’-phosphoribosyl)anthranilate (PRA) from 5-phospho-α-ribose-1-diphosphate (PRPP) and anthranilate and plays an important role in the synthesis of an essential amino acid in M.tuberculosis. A strain with a genetic knockout of the trpD gene, which encodes for the AnPRT enzyme, was unable to cause disease, even in immune-deficient mice. Therefore, this enzyme is a potential drug target for the development of new treatments against TB and other infectious diseases. This research explores the synthesis of different substrates and potential transition state analogues in order to understand catalysis and inhibition of AnPRT enzymes to aid novel drug design. The first part of this study utilises “bianthranilate-like” phosphonate inhibitors that display effective inhibition of the AnPRT enzyme, with the lowest Ki value being 1.3 μM. It was found strong enzymatic inhibition increases with an increased length of the phosphonate linker that occupies multiple anthranilate binding sites within the anthranilate binding channel of the enzyme. Crystal studies of the enzyme in complex with the inhibitors were carried out in order to expose the binding interactions. The second part of this study investigates several new compounds that target the active site of M. tuberculosis AnPRT, based on a virtual screening approach. This approach identified a strong AnPRT inhibitor, which displays an apparent Ki value of 7.0 ± 0.4 μM with respect to both substrates. This study also exposed a conformational change at the active site of the enzyme that occurs on inhibitor binding. The observed conformational changes of the enzyme active site diminish the binding of the substrate PRPP. These pieces of information provide future inhibitor design strategies to aid the development of novel anti-TB agents that target the AnPRT enzyme. To elucidate the reaction mechanism of M. tuberculosis AnPRT, the third part of this study explores the substrate binding sites in detail. This study uses structural analysis, complemented by differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), to reveal detailed information of the substrate and inhibitor binding sites. The final part of this thesis presents the synthesis of various PRPP analogues and potential transition state mimics that were designed based on the likely reaction mechanism of the enzyme. This set of inhibitors includes a number of iminoribitol analogues that were developed to capture the geometry of the flattened ribose ring and include a nitrogen atom within the ring to mimic the positive charge characteristics that are expected in the oxocarbenium-ion-like transition state predicted for M. tuberculosis AnPRT. Additionally, we were able to solve the structure of M. tuberculosis AnPRT in complex with one of the potential transition state mimics, which was observed to bind at the active site of the enzyme. This structure provides new insight into the catalytic mechanism of the enzyme and creates an opportunity to develop more specific inhibitors against the M. tuberculosis AnPRT enzyme.</p>


2021 ◽  
Author(s):  
◽  
Preeti Kundu

<p>Tuberculosis (TB), which is estimated to affect 2 billion individuals worldwide, is an infection predominately caused by Mycobacterium tuberculosis(M. tuberculosis). Of particular concern is the increasing prevalence of TB, which is becoming resistant to the treatments currently available. Anthranilate phosphoribosyltransferase (AnPRT) catalyses the formation of N-(5’-phosphoribosyl)anthranilate (PRA) from 5-phospho-α-ribose-1-diphosphate (PRPP) and anthranilate and plays an important role in the synthesis of an essential amino acid in M.tuberculosis. A strain with a genetic knockout of the trpD gene, which encodes for the AnPRT enzyme, was unable to cause disease, even in immune-deficient mice. Therefore, this enzyme is a potential drug target for the development of new treatments against TB and other infectious diseases. This research explores the synthesis of different substrates and potential transition state analogues in order to understand catalysis and inhibition of AnPRT enzymes to aid novel drug design. The first part of this study utilises “bianthranilate-like” phosphonate inhibitors that display effective inhibition of the AnPRT enzyme, with the lowest Ki value being 1.3 μM. It was found strong enzymatic inhibition increases with an increased length of the phosphonate linker that occupies multiple anthranilate binding sites within the anthranilate binding channel of the enzyme. Crystal studies of the enzyme in complex with the inhibitors were carried out in order to expose the binding interactions. The second part of this study investigates several new compounds that target the active site of M. tuberculosis AnPRT, based on a virtual screening approach. This approach identified a strong AnPRT inhibitor, which displays an apparent Ki value of 7.0 ± 0.4 μM with respect to both substrates. This study also exposed a conformational change at the active site of the enzyme that occurs on inhibitor binding. The observed conformational changes of the enzyme active site diminish the binding of the substrate PRPP. These pieces of information provide future inhibitor design strategies to aid the development of novel anti-TB agents that target the AnPRT enzyme. To elucidate the reaction mechanism of M. tuberculosis AnPRT, the third part of this study explores the substrate binding sites in detail. This study uses structural analysis, complemented by differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC), to reveal detailed information of the substrate and inhibitor binding sites. The final part of this thesis presents the synthesis of various PRPP analogues and potential transition state mimics that were designed based on the likely reaction mechanism of the enzyme. This set of inhibitors includes a number of iminoribitol analogues that were developed to capture the geometry of the flattened ribose ring and include a nitrogen atom within the ring to mimic the positive charge characteristics that are expected in the oxocarbenium-ion-like transition state predicted for M. tuberculosis AnPRT. Additionally, we were able to solve the structure of M. tuberculosis AnPRT in complex with one of the potential transition state mimics, which was observed to bind at the active site of the enzyme. This structure provides new insight into the catalytic mechanism of the enzyme and creates an opportunity to develop more specific inhibitors against the M. tuberculosis AnPRT enzyme.</p>


1994 ◽  
Vol 269 (10) ◽  
pp. 7387-7389
Author(s):  
H. Takei ◽  
Y. Gat ◽  
Z. Rothman ◽  
A. Lewis ◽  
M. Sheves

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