Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of
Structure of the E. coli agmatinase, SPEB
Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E . coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P 3 1 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/R free values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.
more »
« less
- NSF-PAR ID:
- 10275324
- Editor(s):
- Permyakov, Eugene A.
- Date Published:
- Journal Name:
- PLOS ONE
- Volume:
- 16
- Issue:
- 4
- ISSN:
- 1932-6203
- Page Range / eLocation ID:
- e0248991
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+and Mn2+are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+is different and more robust to perturbation than for transporting the toxic Cd2+metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps. -
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
-
Metal ions can play a significant role in a variety of important functions in protein systems including cofactor for catalysis, protein folding, assembly, structural stability and conformational change. In the present work, we examined the influence of alkali (Na, K and Cs), alkaline earth (Mg and Ca) and transition (Co, Ni and Zn) metal ions on the conformational space and analytical separation of mechanically interlocked lasso peptides. Syanodin I, sphingonodin I, caulonodin III and microcin J25, selected as models of lasso peptides, and their respective branched-cyclic topoisomers were submitted to native nESI trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high mobility resolving power of TIMS permitted to group conformational families regardless of the metal ion. The lower diversity of conformational families for syanodin I as compared to the other lasso peptides supports that syanodin I probably forms tighter binding interactions with metal ions limiting their conformational space in the gas-phase. Conversely, the higher diversity of conformational families for the branched-cyclic topologies further supports that the metal ions probably interact with a higher number of electronegative groups arising from the fully unconstraint C-terminal part. A correlation between the lengths of the loop and the C-terminal tail with the conformational space of lasso peptides becomes apparent upon addition of metal ions. It was shown that the threaded C-terminal region in lasso peptides allows only for distinct interactions of the metal ion with either residues in the loop or tail region. This limits the size of the interacting region and apparently leads to a bias of metal ion binding in either the loop or tail region, depending whichever section is larger in the respective lasso peptide. For branched-cyclic peptides, the non-restricted C-terminal tail allows metal coordination by residues throughout this region, which can result in gas-phase structures that are sometimes even more compact than the lasso peptides. The high TIMS resolution also resulted in the separation of almost all lasso and branched-cyclic topoisomer metal ions ( r ∼ 2.1 on average). It is also shown that the metal incorporation ( e.g. , doubly cesiated species) can lead to the formation of a simplified IMS pattern (or preferential conformers), which results in baseline analytical separation and discrimination between lasso and branched-cyclic topologies using TIMS-MS.more » « less
-
The biological importance of lanthanides, and the early lanthanides (La 3+ –Nd 3+ ) in particular, has only recently been recognized, and the structural principles underlying selective binding of lanthanide ions in biology are not yet well established. Lanmodulin (LanM) is a novel protein that displays unprecedented affinity and selectivity for lanthanides over most other metal ions, with an uncommon preference for the early lanthanides. Its utilization of EF-hand motifs to bind lanthanides, rather than the Ca 2+ typically recognized by these motifs in other proteins, has led it to be used as a model system to understand selective lanthanide recognition. Two-dimensional infrared (2D IR) spectroscopy combined with molecular dynamics simulations were used to investigate LanM's selectivity mechanisms by characterizing local binding site geometries upon coordination of early and late lanthanides as well as calcium. These studies focused on the protein's uniquely conserved proline residues in the second position of each EF-hand binding loop. We found that these prolines constrain the EF-hands for strong coordination of early lanthanides. Substitution of this proline results in a more flexible binding site to accommodate a larger range of ions but also results in less compact coordination geometries and greater disorder within the binding site. Finally, we identify the conserved glycine in the sixth position of each EF-hand as a mediator of local binding site conformation and global secondary structure. Uncovering fundamental structure–function relationships in LanM informs the development of synthetic biology technologies targeting lanthanides in industrial applications.more » « less
-
more » « less
Abstract Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pone.0248991
