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Commentary
Revised

Promiscuous scaffolds in proteins - non-native, non-additive and non-trivial

[version 2; peer review: 1 approved with reservations, 2 not approved]
PUBLISHED 20 Jan 2014
Author details Author details
OPEN PEER REVIEW
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Abstract

Promiscuity, the ability of an enzyme to catalyze diverse activities using the same active site, sets up the stage for the evolution of complex organisms through gene duplication and specialization. The detection of promiscuous motifs is crucial to understand the physiological relevance of a protein, or for any endeavor that intends to rationally modify these latent capabilities to design new proteins under laboratory conditions. We have established a methodology for identifying catalytic residues based on spatial and electrostatic congruence with known active site configurations. Here, we discuss insights gained in several initiatives using our method on different enzymes.

Revised Amendments from Version 1

We have updated our manuscript based on the comments of the reviewers. The changes, in brief, are:

  1. Fixed a missing description in Table 1 .
  2. Added a description of known promiscuous proteins.
  3. Added a small paragraph on the related topic of broad specificity.
  4. Minor rephrasing of sentences.

We emphasize once again that this commentary just focuses on results obtained by our group, and is not to be considered as a review.

See the authors' detailed response to the review by Neil D. Rawlings
See the authors' detailed response to the review by Abhinav Nath

Introduction

Primitive life presumably had minimal gene content and a minuscule arsenal of enzymes at its disposal. Unfettered from selection pressures by gene duplication, a few select enzymes gained new advantageous functions13. Nonetheless, the vestiges of secondary activities under neutral drift4 possess the potential to reemerge under changing selection pressures5,6. This ability of an enzyme to catalyze diverse activities using the same active site, termed as promiscuity, is the cornerstone of the evolution of complex organisms from pristine life7. In the human context, compound promiscuity plays a major role in drug discovery, and in the therapeutic efficacy of drugs8. Databases are a crucial medium of cataloging known aspects of drug promiscuity9,10.

Ever since Jensen emphasized the role of promiscuity or ‘substrate ambiguity’ in evolution through ‘fortuitous error and gain of multistep pathways’, promiscuity in proteins has been the subject of intense and detailed research7. It was demonstrated in 1976 that replacing the zinc metal ion by copper in Carboxypeptidase A introduced oxidase catalysis properties11. Dioxygenases promiscuously hydrolyse esters12, while the enolase superfamily is also known to catalyze numerous catalytic reactions13,14. Alkaline phosphatases (AP), one of the key proteins in our research, are one of the most widely researched promiscuous enzymes15. APs are known to have sulfate monoesterase, phosphate diesterase, and phosphonate monoesterase activities1618. A phosphite-dependent hydrogenase activity was also found in Escherichia coli AP (ECAP), but was absent in APs from other organisms19. Interestingly, proteins from the AP superfamily show cross activity - Pseudomonas aeruginosa arylsulfatase (PAS) which has the primary activity of hydrolyzing sulfate monoesters also catalyzes the hydrolysis of phosphate monoesters20,21.

The evolution of species through sequence mutations leaves a trail via the conservation of fragments or repeats that have been honed to achieve specific functions with remarkable efficiency2224. The sequence-to-structure-to-function paradigm facilitates the functional characterization of new proteins by applying a ‘guilt by association’ logic, and has essentially revolutionized the field by its easy to use model25. However, occasionally nature achieves the same solution to an enzymatic problem through a completely different sequence, arriving at the same spatial conformation required for catalysis. For example, the catalytic Ser-His-Asp triad has virtually the same geometry in the major families of serine proteases (chymotrypsin and subtilisin), which have no sequence or structural homology26 - a classical example of convergent evolution27,28. Such convergently evolved proteins, and those redesigned from chiseled scaffolds through exon shuffling, remain beyond the scope of sequence analysis methods. As such structure-based methods have evolved to detect such relationships29,30. The choice of methods for binding site comparisons and methods for binding site detection as well as function prediction has been recently reviewed in detail31. Notably, most of these methods are based on structural properties of the binding or the active site. We have demonstrated that such a structural conservation leading to the same function necessitates the conservation of electrostatic properties as well (CLASP - www.sanchak.com/clasp)32. The ability of finite difference methods to quickly obtain consistent electrostatic properties from peptide structures provides an invaluable tool for investigating other innate properties of protein structures33. Furthermore, using a database of known active sites in proteins (http://www.ebi.ac.uk/thornton-srv/databases/CSA/34), we have proposed a methodology to quantify promiscuity in a wide range of proteins35.

In an endeavor to establish the validity of the computational predictions made by CLASP, we have undertaken several in vitro initiatives using different enzymes. The results of these experiments have provided several insights regarding promiscuous functions in proteins. Foremost amongst them is corroboration of the intuitive notion that inhibition is inherently simpler to predict than true catalysis. For example, we detected the presence of the serine protease (SPASE) catalytic triad motif (Ser195, His57, Asp102) in alkaline phosphatases (AP) from various organisms using the spatial and electrostatic congruence, and validated this by inhibition of the native phosphatase activity using inhibitors (AEBSF/PMSF)32, known to be active on many serine proteases by reaction with the nucleophilic serine36. However, true SPASE activity was limited to shrimp AP. Recently, the crown domain in the E. coli expressed rat intestinal AP protein was shown to be prone to protease cleavage, which the authors have ascribed to self-cleavage37. Another recent review nicely summarizes the various computational approaches applied to the AP superfamily in order to gain insights into the promiscuous functions observed in proteins belonging to the superfamily15. The therapeutic potential of AP inhibitors has also seen increased interest from medicinal researchers38.

In a similar experiment, we detected a SPASE motif in a phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus using CLASP39. Once again, although we easily established the inhibition of the native activity of PI-PLC using serine protease inhibitors, we struggled to establish proteolysis based on known protease substrates. Fortuitously, we observed protease activity of PI-PLC on UVI31+, a protein under investigation in our group for different reasons40. We thus concluded that one should exert caution before ruling out protease activity in an enzyme since theoretically proteases have a large number of possible substrates due to the possible variation in residues flanking the sissile bond, and the corresponding folds that harbor a recognition site for a particular protease39. Thus, it is possible that we have not found the ideal proteolytic substrate for APs32. We also tested the proteolytic functions and inhibition using protease inhibitors of the non-toxic B. cereus phosphatidylcholine-specific phospholipase C (PC-PLC) and the closely related highly toxic Clostridium perfringens α-toxin (CPA) (which possesses an additional C-terminal domain demonstrated to be responsible for its sphingomyelinase, hemolytic, and lethal activities41,42). CPA and PC-PLC activities on phospholipids were unaffected by the addition of serine protease inhibitors in concurrence with the CLASP analysis which fails to detect a SPASE scaffold in these proteins39. While CPA and PC-PLC did have a metallo-protease motif based on CLASP analysis, and both showed protease activity in vitro, the observed proteolytic activity can be attributed as an artifact of a metallo-protease contamination which is difficult to remove in spite of the purification steps. Inhibition of CPA activity using a metallo-protease inhibitor was tried out, but failed to show any results. Such lack of inhibition by a single compound is not sufficient ground to rule out the existence of a metallo-protease scaffold.

Based on predictions from CLASP, we also demonstrated the inhibition of the native phosphatase activity of a cold active alkaline phosphatase from Vibrio strain G15-21 AP (VAP)43 by a specific β-lactam compound (only imipenem, and not by ertapenem, meropenem, ampicillin or penicillin G)44. CLASP analysis detected a spatial and electrostatic congruence of the active site of a Class B2 CphA metallo-β-lactamase (MBL) from Aeromonas hydrophila45 to the active site of VAP. Several β-lactam compounds failed to inhibit E. coli or shrimp AP, as was expected by the lower congruence indicated by CLASP as compared to VAP. While all APs contain three metal ion binding sites essential for catalysis43, MBLs have either one or two metal binding sites46. It would be interesting to imagine the existence of a protein (possibly evolved from VAP) that is an MBL and requires three metal binding sites.

Another desired aspect in the search of promiscuous motifs is the ability to search for partial scaffolds, as has been implemented in the DECAAF methodology47,48. The search for an elastase-like motif in a plant protein47 led us to the pathogenesis-related protein P14a49. Although the complete motif was missing - stated previously as, ‘While Ser195, His57, and Gly193 from the input motif have a highly matching scaffold in P14a, the spatial position of the elastase Asp102 is close to Asn35 and Ser39 in P14a when the proteins are superimposed based on the matching scaffolds48’ - the structural similarity of the P14a protein to a snake venom protein with a known elastase function50 suggested strongly the possibility of pre-existing elastase functionality, or indicated a fair chance of endowing elastase activity through directed evolution techniques.

Another fascinating aspect of enzymes, although strictly not defined as promiscuity, is their ability to catalyze the reaction of a range of similar substrates of the same class51. We have hypothesized that duplicate residues, each of which results in slightly modified replicas of the active site scaffold, are responsible for the broad substrate specificity of proteins52,53.

It might appear that the presence of a motif like a SPASE catalytic triad in a protein structure is trivial, and one could expect any randomly chosen protein with a large number of residues to have such a structural motif. However, the absence of a spatially congruent SPASE catalytic triad in a reasonably large tyrosine phosphatase CD45 (PDBid: 1YGR, sequence length 610) highlights the fact that the SPASE motif is not present ubiquitously (Table 1). Even the presence of a spatially congruent motif, as in the human translation initiation factor (PDBid: 2E9H), does not imply potential congruence (Table 1).

Table 1. Non-triviality of the potential and spatial congruence of the active site residues in proteins from the serine protease motif.

The serine protease catalytic triad has been taken from a non-psychrophilic trypsin from a cold-adapted fish species (PDBid: 1A0J). The reasonably large tyrosine phosphatase CD45 (PDBid: 1YGR, sequence length 610) does not contain the spatially congruent catalytic triad. Although, a motif spatially congruent to the catalytic triad is present in the human translation initiation factor (PDBid: 2E9H), it lacks electrostatic potential congruence. D = Pairwise distance in Å. PD = Pairwise potential difference. SLen = sequence length. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.

PDBActive site atoms(a,b,c)abacbcSLen
1A0JSER195OG,HIS57NE2,ASP102OD1D
PD
3.3
183.7
7.8
153.2
5.6
-30.4
223
1YGRSER1101OG,HIS1041NE2,ASP1043OD1D
PD
6.7
-385.2
13.1
-341.0
7.1
44.1
610
2E9HSER128OG,HIS117NE2,ASP115OD1D
PD
2.9
-44.3
7.1
191.6
6.8
235.9
197

The biggest challenge in detecting promiscuous motifs is to be able to endow the function using rational steps5456. However, the non-additive nature of active site residues makes this a non-trivial task even when a very close partial match exists57. For example in a catalytic site consisting of n residues, the existence of a congruent n−1 motif does not imply that it is easy or even possible to add another residue in the structure and obtain the n residue motif. This complexity is best exemplified in the failure to induce β-lactamase activity in a penicillin-binding protein (PBP-5) from E. coli58,59 by generating the L153E mutant of this protein, as proposed by our previous analysis47 (unpublished results). Although many directed evolution experiments have tried to enhance deacylation in PBPs60, 61, the catalytic step that β-lactamases use to hydrolyze β-lactams62, very few have been successful. Even the successful attempts have reported low gains in β-lactamase activity (110-fold in60 and 90-fold in61).

In spite of the inherent difficultly in rationally designing proteins, we believe that the fast maturing field of protein structure prediction might soon allow us to quickly iterate over in silico mutations63. A method like CLASP may be used to discriminate the predicted structures in order to select the mutations that achieve the desired congruence with a reference scaffold - setting up the flow to mimic the natural ‘evolutionary walk’ in vitro, and accelerate this ‘random walk’ into a ‘resolute sprint’.

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Chakraborty S, Asgeirsson B, Dutta M et al. Promiscuous scaffolds in proteins - non-native, non-additive and non-trivial [version 2; peer review: 1 approved with reservations, 2 not approved]. F1000Research 2014, 2:260 (https://doi.org/10.12688/f1000research.2-260.v2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
VERSION 2
PUBLISHED 20 Jan 2014
Revised
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Reviewer Report 29 Jul 2014
Christopher Bahl, Department of Biochemistry, University of Washington, Seattle, WA, USA 
Not Approved
VIEWS 56
In this commentary, the authors discuss enzyme promiscuity and the difficulties inherent in predicting and experimentally validating enzymatic activity. As stated by the authors, this commentary is intended to be a discussion of their own research. As such, the authors focus ... Continue reading
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HOW TO CITE THIS REPORT
Bahl C. Reviewer Report For: Promiscuous scaffolds in proteins - non-native, non-additive and non-trivial [version 2; peer review: 1 approved with reservations, 2 not approved]. F1000Research 2014, 2:260 (https://doi.org/10.5256/f1000research.3611.r5486)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Version 1
VERSION 1
PUBLISHED 27 Nov 2013
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Reviewer Report 07 Jan 2014
Abhinav Nath, Department of Molecular Biophysics & Biochemistry, Yale University, New Haven CT, USA 
Approved with Reservations
VIEWS 88
The commentary by Chakraborty et al. focuses on the important topics of understanding and modeling enzyme promiscuity, and raises some intriguing points about the importance of local electrostatic effects (beyond structure alone) on enzyme activity. However, the commentary is quite ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Nath A. Reviewer Report For: Promiscuous scaffolds in proteins - non-native, non-additive and non-trivial [version 2; peer review: 1 approved with reservations, 2 not approved]. F1000Research 2014, 2:260 (https://doi.org/10.5256/f1000research.2917.r2833)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 16 Jan 2014
    Sandeep Chakraborty, Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400 005, India
    16 Jan 2014
    Author Response
    Dear Dr Nath,
     
    We would like to thank you for taking the time to review our manuscript. We have responded in detail to another review by Dr Rawlings, and an updated ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 16 Jan 2014
    Sandeep Chakraborty, Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400 005, India
    16 Jan 2014
    Author Response
    Dear Dr Nath,
     
    We would like to thank you for taking the time to review our manuscript. We have responded in detail to another review by Dr Rawlings, and an updated ... Continue reading
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121
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Reviewer Report 20 Dec 2013
Neil D. Rawlings, Wellcome Trust Genome Campus, The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK 
Not Approved
VIEWS 121
This commentary reads more like a review and only describes work previously performed by the researchers. I have a number of reservations about this previously published work. The authors should present more detail from at least one example where dual ... Continue reading
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Rawlings ND. Reviewer Report For: Promiscuous scaffolds in proteins - non-native, non-additive and non-trivial [version 2; peer review: 1 approved with reservations, 2 not approved]. F1000Research 2014, 2:260 (https://doi.org/10.5256/f1000research.2917.r2636)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 16 Jan 2014
    Sandeep Chakraborty, Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400 005, India
    16 Jan 2014
    Author Response
    Dear Dr Rawlings,
     
    We would like to thank you for taking the time to review our manuscript. We appreciate several incisive and relevant points raised by you, and hope that our ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 16 Jan 2014
    Sandeep Chakraborty, Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400 005, India
    16 Jan 2014
    Author Response
    Dear Dr Rawlings,
     
    We would like to thank you for taking the time to review our manuscript. We appreciate several incisive and relevant points raised by you, and hope that our ... Continue reading

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 27 Nov 2013
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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