Pore Engineering for Enhanced Mass Transport in Encapsulin

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Pore engineering for enhanced mass transport in encapsulin nano-compartments. Elsie Williams, Se Min Jung, Jennifer L. Coffman, and Stefan Lutz ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00295 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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ACS Synthetic Biology

Pore engineering for enhanced mass transport in encapsulin nano-compartments.

Elsie A. Williams, Se Min Jung, Jennifer L. Coffman, Stefan Lutz*.

Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30084, United States.

KEYWORDS encapsulin, nanocompartments, protein engineering

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Encapsulins are robust and engineerable proteins that form hollow, nano-sized, icosahedral capsids, making them attractive vehicles for drug delivery, scaffolds for synthetic bionanoreactors, and artificial organelles. A major limitation of native encapsulins is the small size of pores in the protein shell. At 3Å diameter, these pores impose significant restrictions on the molecular weight and diffusion rate of potential substrates. By redesigning the pore-forming loop region in encapsulin from Thermotoga maritima, we successfully enlarged pore diameter up to an estimated 11Å and increased mass transport rates by 7-fold as measured by lanthanide ion diffusion assay. Our study demonstrates the high tolerance of encapsulin for protein engineering and has created a set of novel, functionally improved scaffolds for applications as bionanoreactors.

INTRODUCTION Encapsulins are 32-kDa proteins that spontaneously self-assemble into homomeric, icosahedral nano-compartments. They share a polyhedral shell with bacterial microcompartments (BMCs) but are smaller and simpler designs, consisting of a single protein building block that assembles into cage-like structures of 60 identical subunits, measuring 25 nm in outer diameter with T-1 symmetry (Fig.1). Structural versatility, robustness and evolvability makes them promising scaffolds for drug delivery, bioreactors, and artificial organelles.1-7 Found natively in prokaryotes and archaea, these protein capsids are believed to sequester and remediate toxic species generated during environmental or metabolic stress as they encapsulate cargo proteins such as putative dyedecolorizing peroxidases or ferritin-like proteins (FLPs).8-9 Efficient encapsulation of these proteins is facilitated by a short, C-terminal localization tag on the cargo protein that directs it to


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an interior surface binding site in the encapsulin subunits.10 First described in Brevibacterium linens 11-12, the UniProt database currently lists approximately 600 homologs.13 By utilizing encapsulins as nano-sized architectural scaffolds, protein engineers can exploit their exceptional stability at elevated temperature and wide pH range, as well as resistance to proteolytic degradation. Furthermore, the C-terminal localization tag is generic and has been shown to readily facilitate loading of exogenous proteins and synthetic molecules.3,

14-16

Nevertheless, a major limitation to potential biocatalytic applications is efficient mass transport across the protein shell. In native encapsulin cages, at least three types of pores have been identified at protein subunit interfaces along the edges and vertices of the nano-compartment.8 These pores measure 3-4Å in diameter, making them suitable for metal ion diffusion but highly restrictive to transfer of even small organic molecules.3 For typical substrates and products of molecular weights ≤800 g/mol, an

Figure 1. Structure of encapsulin nano-compartment. A) Schematic of icosahedral protein shell with superimposed molecular structure of single encapsulin (yellow) as part of a vertex-forming pentameric subunit. B) Close-up of pentameric unit and the central 5-fold pore, formed by a six amino acid loop region E184-P189. estimated minimal pore size of ~10Å is necessary to ensure effective passage through the protein shell structure. This assumption is supported by pore sizes of 8-10Å found in BMCs 17 and viral



capsids 2, 18-19, as well as 11Å for diffusion pores in porins such as OmpF 20 and gap junctions 21, all known for effective transport for similar-sized metabolites. The present work focuses on a six amino-acid loop region of encapsulin from Thermotoga maritima (UniProt ID G4FD39; positions 184-189) that shapes a well-defined five-fold pore located at the nano-compartment’s vertices (Fig.1B). Specifically, we have explored the loop region’s tolerance for amino acid substitutions and deletions, and consequently its impact on mass transport across the protein shell. Phylogenetic analysis of encapsulin family members indicates a low degree of sequence conservation in the first three residues, followed by a steady increase to up to 95% for P189 (Fig.S1). Residue conservation suggests functional importance, prompting us to initially conduct alanine-scanning mutagenesis to assess each loop position’s structural role without significantly enlarging the pore itself. Our experiments demonstrated that, despite a significant degree of evolutionary conservation of Y188 and P189, Ala substitutions in each of the six positions (AlaSCAN1-5; Fig.2) were well tolerated and did not result in detectable changes in encapsulin expression levels, self-assembly and capsid stability. Similarly, amino acid replacement of all loop residues (AlaALL) did not affect protein yield and TEM analysis verified the structural integrity of the encapsulin variant (Fig.S2).


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Figure 1. Summary of encapsulin variants with amino acid changes and deletions in pore-forming loop region (bold). Asterisks mark estimated pore diameters.

Given the loop’s amenability to protein engineering, we next explored the effects of systematic deletion of 2 to 8 amino acids in the loop region and beyond. Pore diameters in these variants were estimated to increase from 5Å to 12Å, thereby matching in size the openings found in BMCs and viral capsids. Using AlaALL as template, a series of ∆6 and ∆9 variants were prepared (Fig. 2). Flanking Ala residues in ∆6 variants and Gly substitutions in ∆9 variants proved critical to compensate for steric strain in these truncated encapsulins. Among the ∆6 variants, deletion of two and four loop residues in ∆6-Ala4 and ∆6-Ala2, respectively, were well tolerated as reflected in wild type-like protein expression and intact capsids based on TEM (Fig.3A). In contrast, deletion of the entire six-amino acid stretch (∆6) resulted in low protein yields and showed only partial capsule formation in TEM images. These findings suggest that elimination of the entire loop without compensatory amino acid changes results in unfavorable steric constraints, interfering with effective protein folding and self-assembly. To further test the idea of restricted conformational flexibility, we designed and tested two additional variants (∆9-Gly and ∆9-Gly2) with truncations extending into the flanking helical regions of the encapsulin and one or two Gly residues to relax steric constraints. While ∆9-Gly exhibited similar poor structural behavior as ∆6, ∆9-Gly2 once again yielded robust and completely assembled nanocompartments (Fig.3A). Computational models of ∆6-Ala2 and ∆9-Gly2 based on the T. maritima crystal structure (PDB access: 3dkt)8 estimate enlarged pore diameters of 6 and 11Å, respectively.



Figure 2. Analysis of native and selected encapsulin variants, A) TEM images (scale bar: 25 nm), B) Demonstration of capsid integrity by protease- resistance of free versus encapsulated GCaMP.

To experimentally verify the larger pore diameter of our encapsulin variants, we developed an ion diffusion assay to measure mass transport across the protein shell. In initial tests, native encapsulin and selected variants were coexpressed with GCaMP as cargo protein. GCaMP is a 48 kDa Ca2+-dependent GFP variant consisting of a calmodulin domain inserted into a circular permutated GFP.22 Following the rapid mixing of CaCl2 solution and encapsulated GCaMP sample, the rate of calcium influx into the encapsulin should depend on the size of the pores. Consequently, larger pore diameters will translate into faster development of a fluorescence signal as detected by stopped-flow spectroscopy. For assembly of the encapsulated Ca2+-biosensor, GCaMP was modified with the C-terminal localization tag. As previously shown for other exogenous cargo proteins, encapsulation effectively protects GCaMP from proteolytic degradation (Figs.3B & S3). Trypsin treatment during purification of nano-compartments also ensured removal


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of any contaminating, non-encapsulated biosensor. Following dialysis with EGTA, the Ca-free samples of wild type encapsulin, ∆6-Ala2 and ∆9-Gly2 were tested against free GCaMP in rapid mixing experiments. As expected, the encapsulated biosensor showed significantly slower development of the fluorescence signal compared to free GCaMP. However, rate differences between individual encapsulin variants were within the margins of error (Fig. S4). We suspect that molecular crowding of encapsulated GCaMP interferes with conformational changes of calmodulin upon Ca-binding, making it the rate-determining step in fluorescence onset instead of ion diffusion. Our rationale is supported by independent experiments testing the thermostability of encapsulated GCaMP. These studies showed significant improvements in fluorescence signal retention of encapsulated versus free protein (data not shown). In conclusion, GCaMP is not a suitable sensor for our ion diffusion assay but offers an interesting model system to demonstrate encapsulins' promise as a generic scaffold for improving the thermostability of cargo protein, as well as to mimic cellular crowding in protein folding studies. A lanthanide binding tag (LBT) offers an alternative to GCaMP as a highly sensitive fluorescent ion-biosensor. The short, 17 amino-acid peptide sequence exhibits a characteristic shift in luminescence upon association with selected lanthanide ions.23 The LBT’s small size minimizes possible signal interfere due to conformational changes and simplifies incorporation by being tolerated as an N or C-terminal fusion tag on native and engineered encapsulins (Fig.S5). With the N-terminus of encapsulin located on the interior of the capsid and its C-terminus facing the exterior surface, the position of the LBT tag can easily be controlled. As for the GCaMP study, we built and tested wild type encapsulin, ∆6Ala2, and ∆9Gly2 (all tagged with an N-terminal LBT (NLBT)) in our ion diffusion assay, using TbCl3 as our probe. Wild type encapsulin with a C-terminal LBT (CLBT) served as positive control. Analysis of the tagged encapsulins by stopped-flow



spectroscopy revealed a strong correlation of luminescence signal development with pore diameter (Fig.4). While encapsulin-CLBT showed the expected rapid response, reaching half maximum

Figure 3. Measuring Tb3+ diffusion as a function of encapsulin pore diameter via luminescence build-up in capsids with lanthanide-binding tags by stopped-flow spectroscopy.

signal intensity within an estimated 3.3 ms, the initial rate of luminescence signal of the NLBT in native encapsulin (3Å pore diameter) dropped about 46-fold (Table 1). Doubling the pore diameter to about 6Å in ∆6Ala2 increased the rate of luminescence signal build-up by ~4-fold over wild type. The estimated 11Å pore diameter in ∆9Gly2 raised the rate by another 1.5-fold (or ~6-fold over wild type). While these initial rate estimates are consistent with faster ion diffusion due to increased pore diameter, the fold-changes probably represent low estimates for the actual rate enhancements as approximately half of signal development occurs within the dead time of the stopped-flow instrument. The magnitude of these rate changes as a function of pore diameter correlated well with predicted values based on Fick’s first law of diffusion. Assuming irreversible binding of Tb3+ to the LBT under our experimental conditions (KD = 57 nM,23 100 µM TbCl3), we plotted the inverse values of the measured initial rates of luminescence (Table 1) for the various encapsulin variants against their estimated pore areas. In this simple model, the data points for the


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native NLBT-Encap, NLBT-∆6Ala2, NLBT-∆9Gly2 and Encap-CLBT (pore diameter = ∞) could be fitted by linear equation (r2=0.92).

Table 1. Luminescence (LU) parameters from stopped-flow measurements. max. LU

half max. LU

initial LU rate

(AU)

(ms)

(AU/s-1)

NLB-Encap

2.01 ± 0.01

152 ± 2 (1.0)

13.3 ± 0.2 (1.0)

NLB-∆6Ala2

2.27 ± 0.01

44 ± 1 (3.8)

51 ± 1 (3.8)

NLB-∆9Gly2

1.85 ± 0.01

22 ± 1 (6.9)

83 ± 2 (6.2)

Encap-CLB

2.13 ± 0.02

3.3 ± 0.2 (46)

653 ± 39 (49)

In summary, we have explored by protein engineering the six amino acid loop region which constitutes one of the native pores in the vertex of encapsulin-based nano-compartments. Our study demonstrates that the loop and its flanking helical regions is highly tolerant to amino acid substitutions and deletions of up to 7 residues without interfering with the structural integrity of the protein capsid structure. These alterations enlarge the pore diameter of these nanocompartments up to an estimated 11Å, similar to pore sizes found in viral capsid proteins and gap junctions that effectively transport small molecules and metabolites. These engineered encapsulins hence become attractive scaffolds for further design of robust, nano-scale bioreactors, allowing for more effective mass transport to and from encapsulated chemo or bio-catalysts. Beyond the established encapsulation of catalysts, functionalization of our engineered nano-compartments’ exterior surface offers additional opportunities for building orderly multi-dimensional



architectures and supermacromolecular assemblies involving multiple catalysts for two or threestep reaction cascades.

ASSOCIATED CONTENT Supporting Information. Experimental details on cloning and protein expression/purification, TEM images, data on stopped-flow measurements. This material is available free of charge via the Internet at http://pubs.arc.org.

AUTHOR INFORMATION Corresponding Author [email protected] Funding Sources This work was in part funded by grants from the Emory University Research Committee and the U.S. National Science Foundation (CBET-1706891).

ACKNOWLEDGMENT The authors thank Prof. Robert Campbell for advice on GCaMPs, Matt Jenkins for providing pMATT2, Samantha Iamurri for assistance with the stopped-flow instrument, and all members of the Lutz lab for their helpful comments and suggestions during the preparation of this manuscript.

REFERENCES


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Pore Engineering for Enhanced Mass Transport in Encapsulin

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