Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1

Milić, Dalibor; Dick, Markus; Mulnaes, Daniel; Pfleger, Christopher; Kinnen, Anna; Gohlke, Holger; Groth, Georg

doi:10.1038/s41598-018-21952-3

Cited by 29 publications

(39 citation statements)

References 77 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The criteria 115 for choosing EHs of the flexibility data set are explained in section 3.1. 116 117 118 Comparative models of the flexibility data set (see section 2.1) were generated using our in-119 house structure prediction meta-tool TopModel (26) that has been successfully applied in 120 previous studies (27)(28)(29)(30). TopModel uses multiple state-of-the-art threading and 121 sequence/structure alignment tools to generate a large ensemble of models from different 122 pairwise and multiple alignments of the top five highest ranked template structures.…”

mentioning

confidence: 99%

Promiscuous esterases counterintuitively are less flexible than specific ones

Nutschel

Coscolín

Mulnaes

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

34Understanding mechanisms of promiscuity is increasingly important from a fundamental and 35 application point of view. As to enzyme structural dynamics, more promiscuous enzymes 36 generally have been recognized to also be more flexible. However, examples for the opposite 37 received much less attention. Here, we exploit comprehensive experimental information on 38 the substrate promiscuity of 147 esterases tested against 96 esters together with 39 computationally efficient rigidity analyses to understand the molecular origin of the observed 40 promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly 41 less flexible than specific ones, are significantly more thermostable, and have a significantly 42 increased specific activity. These results may be reconciled with a model according to which 43 structural flexibility in the case of specific esterases serves for conformational proofreading. 44 Our results signify that esterase sequence space can be screened by rigidity analyses for 45 promiscuous esterases as starting points for further exploration in biotechnology and synthetic 46 chemistry. 47 48 3 1. Introduction 49 Enzymes involved in primary metabolism typically exquisitely discriminate against other 50metabolites. Yet, evolution of specificity is only pushed by nature to the point at which 51 'unauthorized' reactions do not impair the fitness of the organism (1). As a result, the universe 52 of promiscuous activities available in nature has been suggested to be enormous (2,3). 53 Understanding mechanisms of promiscuity has thus become increasingly important for the 54 fundamental understanding of molecular recognition and how enzyme function has evolved 55 over time(4) but also to optimize enzyme engineering applications (5). A particular challenge 56 in the latter case is the ability to discover a suitable enzyme with 'sufficient' promiscuous 57 activity to serve as a starting point for further exploration (1). 58Enzyme structural dynamics, besides its role in catalysis (6, 7) and allosteric regulation (8-59 11), has been recognized as likely the single most important mechanism by which promiscuity 60 can be achieved (5). Prominent examples are human cytochrome P450 (CYP) enzymes, for 61 which crystallographic studies and molecular simulations demonstrated that more 62 promiscuous CYPs show larger structural plasticity and mobility (12-14), or TEM-1 -63 lactamase and a resurrected progenitor, for which molecular simulations show that the pocket 64 of the ancestral, and more promiscuous, enzyme fluctuates to a greater extent (15). However, 65 examples for the opposite, i.e., conformational changes selected in evolution such that they 66 enhance specificity in molecular recognition (16), have received much less attention in the 67 context of enzyme promiscuity. 68A clear limitation for scrutinizing the link between enzyme structural dynamics and substrate 69 promiscuity is the general lack of large-scale data on one enzyme (super)family tested against 70 a multitude of ...

show abstract

mentioning

confidence: 99%

Promiscuous esterases counterintuitively are less flexible than specific ones

Nutschel

Coscolín

Mulnaes

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…As a template structure, we used the structure of the B. glumae foldase-lipase complex (PDB code 2ES4) (see Materials and Methods section for details). The final model was assessed with our in-house model quality assessment program TopScore 24,25 and found to be 68% correct for LipA and 52% correct for Lif (Fig. 6E).…”

Section: Resultsmentioning

confidence: 99%

“…To detect changes in sLif:LipA rigidity and flexibility upon Y99A mutation in P. aeruginosa Lif, we analyzed an ensemble of snapshots of sLif-bound LipA in terms of a perturbation approach 26 in a similar way as done by Milić et al 25 . In short, first, an ensemble of 7,500 constraint network topologies was generated from MD snapshots of the proteins sampled at 2 ns intervals from the 10 MD simulations of the sLif:LipA complex (see above).…”

Section: Constraint Network Analysis (Cna)mentioning

confidence: 99%

“…After ten rounds of energy minimization, the C α atoms of the models were superimposed on the template structure and the model with the lowest RMSD was taken for further studies 27 . The model obtained was evaluated by using our in-house model quality assessment program TopScore 24,25 . The correctness of the model is measured as the predicted global and local lDDT score 43 compared to the native structure.…”

mentioning

confidence: 99%

“…(E) Homology model of the P. aeruginosa sLif:LipA complex based on the structure of the B. glumae foldase-lipase complex (PDB code 2ES4 10 ) used as a template. The coloring indicates the model quality assessment by TopScore24,25 , with bluish colors representing less than 10% structural error. (F) CNA was (2020) 10:3578 | https://doi.org/10.1038/s41598-020-60093-4…”

mentioning

confidence: 99%

See 2 more Smart Citations

Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation

Viegas

Dollinger

Verma

et al. 2020

Sci Rep

Self Cite

View full text Add to dashboard Cite

Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K D = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLif Y99A , in which the evolutionary conserved amino acid Y99 from helix α1 of MD1 is mutated to alanine. This coincides with an approximately threefold reduced affinity of the variant to LipA together with increased flexibility of sLif Y99A in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1 Y99A revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLif Y99A. Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones. The Gram-negative human pathogen Pseudomonas aeruginosa produces a wide range of extracellular enzymes 1,2 , among them the lipase LipA, a secreted putative virulence factor 3-5. For its conversion into an enzymatically active conformation, LipA requires the assistance of an inner membrane-bound chaperone named lipase-specific foldase (Lif) 6. On the folding pathway, LipA can adopt several structurally different intermediates: an inactive and unfolded molten globule-like conformation 7 , a near-natively folded pre-active conformation 8 and two folded conformations that differ in the structure of the α-helical lid covering the active site, with the folded closed

show abstract

The Membrane‐Integrated Steric Chaperone Lif Facilitates Active Site Opening of Pseudomonas aeruginosa Lipase A

et al. 2019

Self Cite

View full text Add to dashboard Cite

Lipases are essential and widely used biocatalysts. Hence, the production of lipases requires a detailed understanding of the molecular mechanism of its folding and secretion. Lipase A from Pseudomonas aeruginosa, PaLipA, constitutes a prominent example that has additional relevance because of its role as a virulence factor in many diseases. PaLipA requires the assistance of a membrane-integrated steric chaperone, the lipasespecific foldase Lif, to achieve its enzymatically active state. However, the molecular mechanism of how Lif activates its cognate lipase has remained elusive. Here, we show by molecular dynamics simulations at the atomistic level and potential of mean force computations that Lif catalyzes the activation process of PaLipA by structurally stabilizing an intermediate PaLipA conformation, particularly a β-sheet in the region of residues 17-30, such that the opening of PaLipA's lid domain is facilitated. This opening allows substrate access to PaLipA's catalytic site. A surprising and so far not fully understood aspect of our study is that the open state of PaLipA is unstable compared to the closed one according to our computational and in vitro biochemical results. We thus speculate that further interactions of PaLipA with the Xcp secretion machinery and/or components of the extracellular matrix contribute to the remaining activity of secreted PaLipA.

show abstract

Recognition motif and mechanism of ripening inhibitory peptides in plant hormone receptor ETR1

Cited by 29 publications

References 77 publications

Promiscuous esterases counterintuitively are less flexible than specific ones

Promiscuous esterases counterintuitively are less flexible than specific ones

Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation

The Membrane‐Integrated Steric Chaperone Lif Facilitates Active Site Opening of Pseudomonas aeruginosa Lipase A

Contact Info

Product

Resources

About