A 2.13 Å Structure of <i>E. coli</i> Dihydrofolate Reductase Bound to a Novel Competitive Inhibitor Reveals a New Binding Surface Involving the M20 Loop Region

Summerfield, R.; Daigle, Denis M.; Mayer, Stanislas; Mallik, Debasis; Hughes, Donald W.; Jackson, S.G.; Sulek, M.; Organ, Michael G.; Brown, Eric D.; Junop, M.S.

doi:10.1021/jm060570v

Cited by 45 publications

(37 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the phenformin complex, the N1-subsituted nitrogen interacts directly with Asp27, positioning the phenethyl sidechain for interactions with Leu28 and, more weakly, with Phe31 and Ile50. The binding site orientation in the buformin complex is more similar to related structures previously reported (pdb: 2ANQ 65 ; pdb: 3DGA 25 ). The PFM complex also exhibits a uniquely positioned Met20 loop, in which Met16 is recruited into the active site where it interacts with the phenyl ring of the inhibitor (Figure S1).…”

Section: Resultssupporting

confidence: 84%

A Structural Basis for Biguanide Activity

et al. 2017

View full text Add to dashboard Cite

Metformin is the most commonly prescribed treatment for Type II diabetes and related disorders, however molecular insights into its mode(s) of action have been limited by an absence of structural data. Structural considerations along with an increasing body of literature demonstrating its effects on one-carbon metabolism suggest the possibility of folate mimicry and anti-folate activity. Motivated by increasing recognition that anti-diabetic biguanides may act directly upon the gut microbiome, we have determined structures of the complexes formed between the anti-diabetic biguanides: phenformin, buformin, and metformin and E. coli dihydrofolate reductase (ecDHFR) based on NMR, crystallographic and molecular modeling studies. Inter-ligand Overhauser effects indicate that metformin can form ternary complexes with p-aminobenzoyl-L-glutamate (pABG) as well as other ligands that occupy the region of the folate binding site that interacts with pABG, however DHFR inhibition is not cooperative. The biguanides inhibit the activity of ecDHFR competitively, with the phenformin inhibition constant 100-fold lower than that of metformin. This inhibition may be significant at concentrations present in the gut of treated individuals, and inhibition of DHFR in intestinal mucosal cells may also occur if accumulated levels are sufficient. Perturbation of folate homeostasis can alter the pyridine nucleotide redox ratios that are important regulators of cellular metabolism.

show abstract

Section: Resultssupporting

confidence: 84%

A Structural Basis for Biguanide Activity

et al. 2017

View full text Add to dashboard Cite

show abstract

“…against ecDHFR revealed two novel inhibitors that have a guanidine in place of the diaminopyrimidine ring structure [25]. Structural data for compound C1A (figure 8) reveals that the primary interaction of the isothiourea of C1A is with Asp27, similar to MTX, while the other isothiourea moiety binds in a pocket that is not normally occupied by those inhibitors reported in table 5 [95] (figure 15a). Data were also reported for a less potent compound, 817 (figure 8), that reveals binding of the isothiourea to Asp27 and further shows that the 3 0 -trifloro ring has a rotational disorder, making it equivalent to a 3 0 ,5 0 -disubstituted ring (figure 15b).…”

Section: Othermentioning

confidence: 99%

Structural characteristics of antifolate dihydrofolate reductase enzyme interactions

Cody¹,

Schwalbe²

2006

Crystallography Reviews

View full text Add to dashboard Cite

The ubiquitous enzyme dihydrofolate reductase (DHFR) is responsible for the reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate in an NADPH-dependent manner. It is also a key pharmacological target for the treatment of cancer, as well as bacterial and opportunistic pathogenic infections. Interest in the design of potent and selective antifolate inhibitors has made DHFR one of the most studied enzymes, in particular its structural and biochemical properties. This review surveys more than 129 DHFR solution and crystal structures currently (02/07) reported in the Protein Data Bank representing 15 species of enzyme. Comparison of these DHFR sequences shows that while there is a high sequence homology among vertebrate species (75-95%), there is only about 30% homology between vertebrate and bacterial species. Despite the highly conserved nature of the ligand and cofactor binding sites, DHFR can bind a wide range of compounds that can have a high degree of flexibility. The enzyme itself can also undergo ligand-induced conformational changes that reflect its catalytic mechanism of action. Mechanistic questions can now be addressed with the structural data available for atomic resolution enzyme complexes as well as from neutron diffraction data that have recently become available. These data provide new insight into the design of novel inhibitors that can target specific species with high selectivity of binding.

show abstract

“…Selection of incision sites were based on the wtDHFR structure (PDB code 2ANO)28, which bound both an NADPH and an inhibitor molecule. In some cases, the loops were trimmed but no new residues were added.…”

Section: Methodsmentioning

confidence: 99%

Protein rethreading: A novel approach to protein design

Agah

Poulos

et al. 2016

Sci Rep

View full text Add to dashboard Cite

Protein engineering is an important tool for the design of proteins with novel and desirable features. Templates from the protein databank (PDB) are often used as initial models that can be modified to introduce new properties. We examine whether it is possible to reconnect a protein in a manner that generates a new topology yet preserves its structural integrity. Here, we describe the rethreading of dihydrofolate reductase (DHFR) from E. coli (wtDHFR). The rethreading process involved the removal of three native loops, and the introduction of three new loops with alternate connections. The structure of the rethreaded DHFR (rDHFR-1) was determined to 1.6 Å, demonstrating the success of the rethreading process. Both wtDHFR and rDHFR-1 exhibited similar affinities towards methotrexate. However, rDHFR-1 showed no reducing activity towards dihydrofolate, and exhibited about ~6-fold lower affinity towards NADPH than wtDHFR. This work demonstrates that protein rethreading can be a powerful tool for the design of a large array of proteins with novel structures and topologies, and that by careful rearrangement of a protein sequence, the sequence to structure relationship can be expanded substantially.

show abstract

A 2.13 Å Structure of E. coli Dihydrofolate Reductase Bound to a Novel Competitive Inhibitor Reveals a New Binding Surface Involving the M20 Loop Region

Cited by 45 publications

References 21 publications

A Structural Basis for Biguanide Activity

A Structural Basis for Biguanide Activity

Structural characteristics of antifolate dihydrofolate reductase enzyme interactions

Protein rethreading: A novel approach to protein design

Contact Info

Product

Resources

About