FUN_12

The role of MbtH-like proteins in nonribosomal peptides synthetase (NRPS) assembly-lines: Tools for biosynthesis/bioengineering of novel antibiotics.

THE PROJECT PROPOSAL IS PRESENTED HERE AND THE FINAL REPORT IS PRESENTED BELOW.


SSGCID Functional Project Proposal: 
SSGCID PI/Lead:  Garry W. Buchko
Co-investigator: Sylvie Garneau-Tsodikova, Dept. of Pharmaceutical Sciences, University of Kentucky, Lexington, KY.


Many of the naturally derived antibiotics in current use (along with many other pharmaceuticals such as anti-tumor agents and immuno-suppressants) are complex secondary metabolites biosynthesized on nonribosomal peptide synthetase (NRPS) “assembly-lines” by bacteria.  These assembly-lines contain many proteins usually encoded in gene clusters on the chromosome and comprise multiple modules each containing three core domains for condensation (C), adenylation (A), and thiolation (T).  The most studied systems are from Streptomyces coelicolor, a soil-dwelling filamentous bacteria that contains 18 complex secondary metabolite gene clusters1.  Common to most NRPS gene clusters is a small protein comprised of ~70 amino acids, which plays an important role in stimulating the adenylating activity of A domains in NRPS modules2,3.  Garneau-Tsodikova and co-workers have shown that three such proteins (SAMR0548, KtzJ, and TioT) were essential for the activation of congocidine, kutzneride, and thiocoraline NRPS systems, respectively, of S. coelicolor4-7.  Furthermore, they recently found that co-expression of TioT homologues from other NRPS systems with TioK resulted in a drastic change in the biochemical function of the complex (manuscript in preparation). While the kinetic parameters for the main substrate remained excellent, even in the presence of a non-cognate partners for TioK, the substrate profile of the A domain of the A-T TioK di-domain changed and was broadened.  Thus, by combining a variety of MbtH-like proteins with different NRPS partners, it may be possible to create a toolbox for the bioengineering of novel pharmaceuticals. 

In Mycobacterium tuberculosis, the NRPS assembly-line is responsible for producing two types of siderophores, called mycobactins, responsible for the sequestration of iron and assisting M. tuberculosis survival under the low-iron conditions of human infection.  SSGCID has recently determined the solution structure of three members of this gene family (from M. tuberculosis (MbtH, MytuD.01649, 2KHR), M. murinum (MymaA.01649.c, 2MYY), and M. avium, (Myav.01649.c, soon to be submitted to PDB); as well as Burkholderia pseudomallei, BupsA.13472.b, 2LPD).  All contain a three-strand anti-parallel b-sheet (b3:b1:b2) nestled against one C-terminal a-helix8

The aim of our functional study is to use these MtbH-like proteins to expand the NRPS toolbox and determine the structural basis driving the functional variability.  A structure for M. tuberculosis MbtB has not been determined because, like TioN and TioK, it cannot be expressed in soluble and active form by itself.

Specific Aim 1: Construct heterologous NRPS co-expression systems.

Genes for the MtbH-like proteins from SSGCID and CSGID relevant bacterial pathogens (including those described above) will be co-expressed with the S. albus NRPS proteins TioK and TioN and the co-expressed complexes assayed for a wide range of NRPS activities.  This work will be performed in the Garneau-Tsodikova laboratory at the University of Kentucky.

 

Specific Aim 2: Determine crystal structure of TioK and TioN in complex with various MbtH-like proteins.

Purified co-expressed proteins will be shipped by Garneau-Tsodikova for placement into crystallization screens at Beryllium.  If necessary, additional protein expression and purification will be done by Dr. Buchko at PNNL. 

 

Specific Aim 3:  Determine a crystal structure for a MbtH-MbtB complex and assay activity.

Because S. albus is in the same taxonomic order (Actinomycetales) as the mycobacteria, MbtH will also be co-expressed with the M. tuberculosis protein (MbtB) homologous to TioN.  A structure for M. tuberculosis MbtB has not been determined most likely because, in analogy with TioN and TioK, it cannot be expressed in soluble and active form by itself. Cloning, protein purification, and assay work will be performed in the Garneau-Tsodikova laboratory at the University of Kentucky. Crystallization screens and structure determination will be performed at Beryllium and any additional protein expression and purification needed will be done by Dr. Buchko at PNNL.

TIMELINE

MILESTONE

Quarter 1

09.2015-11.2015

  • M1: Clone MbtH-like proteins into heterologous co-expression systems with TioN and TioK (supports Aim 1)
  • M2: Assay complexes for a suite of activities (supports Aim 1)

Quarter 2

12.2015-02.2016

  • M3: Clone M. tuberculosis MbtH into co-expression systems with M. tuberculosis proteins (starting with MbtB) (supports Aim 3)
  • M4: Purify selected co-complexes and set up crystal screens (supports Aims 2 and 3)

Quarter 3

03.2016-05.2016

  • M5: Solve crystal structures of co-complexes (supports Aims 2 and 3)
  • M6: Potentially solve structures of complexes with substrates identified in the activity assays (supports Aims 2-3)

Quarter 4

06.2016-08.2016

  • M7: Complete any remaining studies (e.g., ITC, NMR-based characterization of catalysis products) to strengthen publications in high impact journals (supports Aims 2-3)
  • M8: Publish results (supports Aims 2-3)

 

Outcomes

Solving structures of multi-protein complexes is “the next big thing” in structural biology and the two-component expression system for NRPS assembly-line proteins designed by Garneau-Tsodikova and co-workers offers a mechanism for SSGCID to get more involved in this game.  Moreover, the ultimate goal of this project, a toolbox for the bioengineering of novel pharmaceuticals, aligns with NIAID’s mission of using structural biology to assist the structure-based design of novel antimicrobial agents.  The activity assays with multiple MbtH-like proteins and TioK and TioN will expand the available bioengineering toolbox and the complementary crystal structures of the MbtH-like proteins with TioK and TioN will provide a structural basis to explain the molecular reactions these complexes perform and the role of MbtH-like proteins in influencing the biochemistry.  Not only will the structures of these MbtH-like proteins with TioK and TioN assist the structure based design of novel biosynthesized antibiotics, the co-expression protocol with MtbH-like proteins may be the only method of determining a structure for M. tuberculosis MbtB and some of the other proteins in the M. tuberculosis NRPS assembly-line that are responsible for producing siderophores.  The structures of these later complexes will assist the structure-based design of antibiotics based on compounds that prevent the formation of these complexes and the consequential iron-starvation of M. tuberculosis following infection.

References

1.  Bentley SD, et al. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature. 417(6885):141-147 (2002)

2.  Felnagle EA, et al. MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases. Biochemistry. 49(41):8815-8817 (2010)

3.  Zhang W, Heemstra JR, Jr., Walsh CT, Imker HJ. Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins. Biochemistry. 49(46):9946-9947 (2010)

4.  Al-Mestarihi AH, Garzan A, Kim JM, Garneau-Tsodikova S. Enzymatic evidence for a revised congocidine biosynthetic pathway. ChemBioChem. (2015) doi: 10.1002/cbic.201402711

5.  Zolova OE, Garneau-Tsodikova S. KtzJ-dependent serine activation and O-methylation by KtzH for kutznerides biosynthesis. J. Antibiotics. 67(1):59-64 (2014)

6.  Zolova OE, Garneau-Tsodikova S. Importance of the MbtH-like protein TioT for production and activation of the thiocoraline adenylation domain of TioK. MedChemComm. 3:950-955 (2012)

7.  Al-Mestarihi AH, Villamizar G, Fernandez J, Zolova OE, Lombo F, Garneau-Tsodikova S. Adenylation and S-methylation of cysteine by the bifunctional enzyme TioN in thiocoraline biosynthesis. J. Am. Chem. Soc.. 136(49):17350-17354 (2014)

8.  Buchko GW, Kim CY, Terwilliger TC, Myler PJ. Solution structure of Rv2377c-founding member of the MbtH-like protein family. Tuberculosis. 90(4):245-251 (2010)

FINAL REPORT

Functional Study 12: MbtH-like proteins.

Project lead: Sylvie Garneau-Tsodikova, University of Kentucky

Co-investigator: Garry Buchko, PNNL

Status: Completed

Time-line: Sept 2015 - August 2016

ORIGINAL

TIMELINE

MILESTONE

ACHIEVED

Quarter 1

09.2015-11.2015

  • M1: Clone MbtH-like proteins into heterologous co-expression systems with TioN and TioK (supports Aim 1)
  • M2: Assay complexes for a suite of activities (supports Aim 1) 

X


 X*

Quarter 2

12.2015-02.2016

  • M3: Clone M. tuberculosis MbtH into co-expression systems with M. tuberculosis proteins, starting with MbtB (supports Aim 3)
  • M4: Assay and purify selected co-complexes and set up crystal screens (supports Aims 2 and 3)

X

 

In progress

Quarter 3

03.2016-05.2016

  • M5: Solve crystal structures of co-complexes (supports Aims 2 and 3)
  • M6: Potentially solve structures of complexes with substrates identified in the activity assays

In progress

 

In progress

Quarter 4

06.2016-08.2016

  • M7: Complete any remaining studies (e.g. ITC, NMR-based characterization of catalysis products) to strengthen publications in high impact journals (supports Aims 2 and 3)
  • M8: Publish results (supports Aims 2 and 3)

In progress

 

In progress

*Work completed for majority of combinations. Some work may continue outside of this functional by the collaborator.

Summary: The primary goal of the functional study is to generate, then biochemically and structurally study MbtH-like proteins in conjunction with adenylation enzymes. During the second half of the functional study the Garneau-Tsodikova lab achieved purification of most of the MbtH-like targets (provided by SSGCID) in complex with partnering adenylation enzymes. Milestones M1, M2 and M3 are complete and since the crystallization trials will continue for some months into the next award year, and since the collaborator intends to continue assay work in her lab, we have indicated that the remaining milestones (M4, M5, M6, M7, M8) are still in progress. 

Specific Aim 1:  Construct heterologous NRPS co-expression systems.

Completed. Summarized in the following updated tables are the current progress of the cloning and sequential transformation (Table 5) for the 9 genes to be combined with TioK or TioN, and the expression and purification of those combinations (Table 6). At this time no further coexpression constructs will be made, thus we have indicated work for this Aim as completed (M1, M2, M3). However there are still some combinations that can be further explored should a specific A-domain/MbtH0like protein combination prove fruitful.  

Table 5. Cloning and sequential transformation of MbtH-like proteins together with TioK or TioN.

Specific Aim 2:  Determine crystal structure of TioK and TioN in complex with various MbtH-like proteins

In progress. Combinations of MbtH-like proteins and TioK or TioN are still on-going and additional results are shown in Table 7. Those checks in Table 6. having a value or note in parentheses are those that have been or attempted to purify. Those with a blue code starting with MispA have been submitted for crystallographic studies. In addition, TioK and TioN with TioT in pACYCDuet-1 with a NHis tag have also been purified and submitted for crystallography. Of particular note, TioK with ButhA.13472.a.B1 in pACYCDuet-1 with no tag and MypaA.01649.a.A1 in pACYCDuet-1 with a NHis tag and TioN with with ButhA.13472.a.B1 in the provided vector and MypaA.01649.a.A1 in pACYCDuet-1 with a NHis tag generated an insoluble complex. TioK combined with ButhA.13472.a.B1 only yielded soluble MbtH-like protein.

To date, all complexes submitted for crystallography have been tested using the inorganic pyrophosphate exchange assay (M4) and to adenylate tryptophan (TioK) and cysteine (TioN) to a greater extent than the phenylalanine (TioK) and serine (TioN) controls. The collaborator intends to continue the functional assay work to examine if the different MbtH-like proteins will affect the kinetics of the substrates and/or expand the substrate profile for the A-domains. 

Since some of the purifications were completed only recently, it is expected that crystallization studies will continue into the coming award year. We have therefore indicated that milestones M5 and M6 are still in progress.

 

Table 6. Protein co-expression and complex purification of MbtH-like proteins together with TioK or TioN.

Purifications indicated by a blue code starting with MispA have been submitted for crystallographic studies.

 

Specific Aim 3:  Determine a crystal structure for a MbtH-MbtB complex and assay activity systems.

Full-length MbtB was successfully amplified from the genomic DNA of Mycobacterium tuberculosis using standard PCR porotocls and inserted into pET28a. The new plasmid was then transformed into the E. coli BL21(DE3) ybdz::aac(3)-iv cell line for the dual expression with MbtH.  The cells were made competent and MbtH was successfully transformed into cells, visualized by dual selection media. Preliminary tests failed to express MbtB. Growth optimization and possible truncation of the A-domain are currently under investigation.

Future Studies/Ongoing Studies: 

There will be one last shipment of 3-4 proteins complexes that will be shipped by the before the end September, 2016 for crystallographic studies. The activity of these enzymes still needs to be determined, but will likely be accomplished by the end September as well. Additional studies will include working the MbtB to express and purify with MbtH and examine its activity. When the last remaining experiments for Specific Aim 2 are completed, the data will be presentable for publication and the effect of these MbtH-like proteins on TioK and TioN will be published. Thus, milestones M7 and M8 are listed as in progress. 


 

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