NMR Structure of Mistic, a Membrane-Integrating Protein

for Membrane Protein Expression

NMR Structure of Mistic, a Membrane-Integrating Protein

for Membrane Protein Expression

By: Niloufar SafvatiBy: Niloufar Safvati

Structure Determination of Membrane Proteins

Structure Determination of Membrane Proteins

Structural determination of soluble proteins has minimal restraints

Structural determination of Membrane Proteins, however, has a couple of restraints:

1. Production of high enough yield of protein

2. Crystallization

Structural determination of soluble proteins has minimal restraints

Structural determination of Membrane Proteins, however, has a couple of restraints:

1. Production of high enough yield of protein

2. Crystallization

Characteristics of an ideal fusion partner that is specialized in producing

recombinant IM proteins

Characteristics of an ideal fusion partner that is specialized in producing

recombinant IM proteins

An ideal fusion partner should:

§ autonomously traffic its cargo to the membrane, bypassing the translocon and associated toxicity issues

§ retain the characteristics of other successful fusion partner proteins, including relatively small size, in vivo folding, and high stability.

An ideal fusion partner should:

§ autonomously traffic its cargo to the membrane, bypassing the translocon and associated toxicity issues

§ retain the characteristics of other successful fusion partner proteins, including relatively small size, in vivo folding, and high stability.

NMR SpectroscopyNMR Spectroscopy

Can be used as an alternative method to crystallization

NMR structure determination of IM proteins has been established only for very small, structurally simplistic IM proteins and for outer membrane bacterial porins

New techniques for determining the characteristics of alpha helical IM proteins are therefore necessary

Can be used as an alternative method to crystallization

NMR structure determination of IM proteins has been established only for very small, structurally simplistic IM proteins and for outer membrane bacterial porins

New techniques for determining the characteristics of alpha helical IM proteins are therefore necessary

What is Mistic?What is Mistic? Mistic is a Bacillus subtilis integral

membrane protein that folds into the membrane without the help of a translocon

Mistic stands for Membrane-Integrating Sequence for Translation of Integral Membrane protein Constructs

It consists of 110-amino acids (13kD)

Mistic is a Bacillus subtilis integral membrane protein that folds into the membrane without the help of a translocon

Mistic stands for Membrane-Integrating Sequence for Translation of Integral Membrane protein Constructs

It consists of 110-amino acids (13kD)

Why study Mistic?Why study Mistic? When recombinantly expressed in E. coli, Mistic

associates tightly with the bacterial membrane. Surprisingly, Mistic is highly hydrophilic Mistic has most of the characterizations for being an ideal

partner in the production of high-yields of integral membrane proteins

When recombinantly expressed in E. coli, Mistic associates tightly with the bacterial membrane.

Surprisingly, Mistic is highly hydrophilic Mistic has most of the characterizations for being an ideal

partner in the production of high-yields of integral membrane proteins

Mistic CharacterizationsMistic Characterizations The in vivo topology of Mistic in E. coli was analyzed by

evaluating the accessibility of an array of monocysteine mutants to the membrane-impermeable thiol biotinylating reagent 3-(N-maleimidopropinyl) biocytin (MPB).

In addition to the single naturally occurring cysteine (residue 3), cysteine mutations were introduced individually at the C terminus (residue 110) and in predicted loop regions at positions 30, 58, and 88, with the naturally occurring cysteine mutated to valine.

Result:This experiment revealed a well- exposed periplasmic C terminus. The lack of reactivity of the other locations indicates that they are either intracellular or membrane-embedded in Mistic’s native conformation.

The in vivo topology of Mistic in E. coli was analyzed by evaluating the accessibility of an array of monocysteine mutants to the membrane-impermeable thiol biotinylating reagent 3-(N-maleimidopropinyl) biocytin (MPB).

In addition to the single naturally occurring cysteine (residue 3), cysteine mutations were introduced individually at the C terminus (residue 110) and in predicted loop regions at positions 30, 58, and 88, with the naturally occurring cysteine mutated to valine.

Result:This experiment revealed a well- exposed periplasmic C terminus. The lack of reactivity of the other locations indicates that they are either intracellular or membrane-embedded in Mistic’s native conformation.

Only Glu110 at the C terminus is well exposed periplasmically

Orange: monocysteine probing residues

Primary sequence of Mistic:

Green: structural disruption mutants

Gray: cloning artifact residues

Secondary Structure of MisticSecondary Structure of Mistic The secondary structure of Mistic was analyzed through NMR

spectroscopy. The primary sequence was given backbone assignments which

includes: 1. The use of Transverse Relaxation Optimized Spectroscopy

(TROSY) 2. The use of Nuclear Overhauser Effect Spectroscopy (NOESY)Result:

The 13Calpha chemical shift deviation from random coil values, the observed NOE pattern, and slow 1HN exchange with solvent strongly indicate the presence of four helices comprising residues 8 to 22, 32 to 55, 67 to 81, and 89 to 102.

The secondary structure of Mistic was analyzed through NMR spectroscopy.

The primary sequence was given backbone assignments which includes:

1. The use of Transverse Relaxation Optimized Spectroscopy (TROSY)

2. The use of Nuclear Overhauser Effect Spectroscopy (NOESY)Result:

The 13Calpha chemical shift deviation from random coil values, the observed NOE pattern, and slow 1HN exchange with solvent strongly indicate the presence of four helices comprising residues 8 to 22, 32 to 55, 67 to 81, and 89 to 102.

•Values larger than 1.5 ppm are indicative of an a-helical secondary structure

•Values smaller than -1.5 ppm are indicative of ß-sheet secondary structure.

Alpha Helices and Beta-sheets

Blue-chemical shifts in 0 mM K+

Green-chemical shifts in 100 mM K+

Transverse relaxation optimized spectroscopy (TROSY)

Transverse relaxation optimized spectroscopy (TROSY)

The NMR signal of large molecules has shorter transverse relaxation times compared to smaller molecules and therefore decays faster, leading to line broadening in the NMR spectrum which gives poor resolution and makes it difficult to analyze the molecule.

The TROSY experiment is designed to choose the component for which the different relaxation mechanisms have almost cancelled, leading to a single, sharp peak in the spectrum. This significantly increases both spectral resolution and sensitivity leading to better results.

The NMR signal of large molecules has shorter transverse relaxation times compared to smaller molecules and therefore decays faster, leading to line broadening in the NMR spectrum which gives poor resolution and makes it difficult to analyze the molecule.

The TROSY experiment is designed to choose the component for which the different relaxation mechanisms have almost cancelled, leading to a single, sharp peak in the spectrum. This significantly increases both spectral resolution and sensitivity leading to better results.

Transverse Relaxation Optimized Spectroscopy (TROSY)

Transverse Relaxation Optimized Spectroscopy (TROSY)

Fernandex and Wider, Current Opinion in Structural Biology 2003, 13:570-580

Nuclear Overhauser Effect Spectroscopy (NOESY)

Nuclear Overhauser Effect Spectroscopy (NOESY)

The Nuclear Overhauser Effect (NOE) is the transfer of nuclear spin polarization from one spin to another and is shown through NMR spectroscopy.

All atoms that are in proximity to each other give a NOE.

The distance can be derived from the observed NOEs, so that the precise, three-dimensional structure of the molecule can be reconstructed.

The Nuclear Overhauser Effect (NOE) is the transfer of nuclear spin polarization from one spin to another and is shown through NMR spectroscopy.

All atoms that are in proximity to each other give a NOE.

The distance can be derived from the observed NOEs, so that the precise, three-dimensional structure of the molecule can be reconstructed.

Folding of MisticFolding of Mistic Unlike the secondary structure determination, long-

range restraints are necessary to determine the fold of the protein

The monocysteine mutant library described in the topology assay was used to incorporate site-directed spin labels within Mistic that produce distance-dependent line- broadening perturbations in the NMR spectra that could be translated into distances for structure determination

The signal changes observed for the five spin-labeled samples were transformed into 197 long-range upper-distance and 290 lower- distance restraints

Unlike the secondary structure determination, long-range restraints are necessary to determine the fold of the protein

The monocysteine mutant library described in the topology assay was used to incorporate site-directed spin labels within Mistic that produce distance-dependent line- broadening perturbations in the NMR spectra that could be translated into distances for structure determination

The signal changes observed for the five spin-labeled samples were transformed into 197 long-range upper-distance and 290 lower- distance restraints

ResultsResults

After collecting all the NOE data, angle restraints, spin labeling restraints and α-helical hydrogen bond restraints, the final structure calculation resulted in:

1. 573 NOE distance restraints2. 346 angle restraints from chemical shifts

and NOEs3. 478 distance restraints from spin-label

experiments

After collecting all the NOE data, angle restraints, spin labeling restraints and α-helical hydrogen bond restraints, the final structure calculation resulted in:

1. 573 NOE distance restraints2. 346 angle restraints from chemical shifts

and NOEs3. 478 distance restraints from spin-label

experiments

3-D Structure of Mistic3-D Structure of Mistic The bundle of 10 conformers with the lowest target

function is used to represent the three-dimensional NMR structure.

The loop connecting α2 and α3, as well as the C terminus of Mistic, are more mobile. (This proves to be important further into the experiment)

The bundle of 10 conformers with the lowest target function is used to represent the three-dimensional NMR structure.

The loop connecting α2 and α3, as well as the C terminus of Mistic, are more mobile. (This proves to be important further into the experiment)

All helices except α2 are slightly shorter than expected for a bilayer- traversing helix

This is likely due to partial unraveling of the ends of the helices in the detergent micelle environment, especially at the N and C termini (α1 and α4) allows Mistic to adapt to the lipid environment

Helix α2 has a kink

All helices except α2 are slightly shorter than expected for a bilayer- traversing helix

This is likely due to partial unraveling of the ends of the helices in the detergent micelle environment, especially at the N and C termini (α1 and α4) allows Mistic to adapt to the lipid environment

Helix α2 has a kink

Surprising Structure of MisticSurprising Structure of Mistic

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are needed to see this picture.

•Mistic appears to have hydrophilic surface for an IM protein even though it is assembled internally with a typical hydrophobic core.

•Given the membrane-traversing topology demonstrated by the MPB labeling experiment this is an unusual surface property.

Confirming The Unusual Hydrophilic Surface

Confirming The Unusual Hydrophilic Surface

NOEs between Mistic and its solubilizing LDAO detergent micelle were measured and assigned.

When sites with NOE signals are mapped to the surface of the Mistic structure, a concentric ring of detergent interactions around the helical bundle is observed, as expected for a membrane-integrated protein.

Results:Mistic is embedded within the LDAO micelle.

NOEs between Mistic and its solubilizing LDAO detergent micelle were measured and assigned.

When sites with NOE signals are mapped to the surface of the Mistic structure, a concentric ring of detergent interactions around the helical bundle is observed, as expected for a membrane-integrated protein.

Results:Mistic is embedded within the LDAO micelle.

Variable ConformationVariable Conformation Mistic might be exploited to target another protein to the bacterial

membrane, when fused to Mistic’s C terminus, such that it too could readily fold into its native, lipid bilayer inserted conformation.

Mistic-assisted expression of three topologically and structurally distinct classes of eukaryotic IM proteins were tested: 1. voltage-gated K+ channels2. receptor serine kinases of the transforming growth factor-ß (TGF-b) superfamily3. G-protein coupled receptors (GPCRs)

Result: In 15 of the 22 tested constructs the desired product could be isolated from the membrane fraction of recombinant bacteria at yields exceeding 1 mg per liter of culture.

Mistic might be exploited to target another protein to the bacterial membrane, when fused to Mistic’s C terminus, such that it too could readily fold into its native, lipid bilayer inserted conformation.

Mistic-assisted expression of three topologically and structurally distinct classes of eukaryotic IM proteins were tested: 1. voltage-gated K+ channels2. receptor serine kinases of the transforming growth factor-ß (TGF-b) superfamily3. G-protein coupled receptors (GPCRs)

Result: In 15 of the 22 tested constructs the desired product could be isolated from the membrane fraction of recombinant bacteria at yields exceeding 1 mg per liter of culture.

Figure B:

The Mistic-fused protein is shown on the left (open arrow)

The final product after removal of Mistic by thrombin digestion is on the right (solid arrow).

Mistic Produces High Yields of IM Proteins

Mistic Produces High Yields of IM Proteins

The identity of the resulting bands are determined by N-terminal sequencing

In addition, aKv1.1 was extracted and purified in LDAO to verify that the protein resembled its native conformation. Gel-filtration showed the structure is a tetramer.

Results: There exists a high propensity for this system to

produce IM proteins fully folded in their native conformations

The identity of the resulting bands are determined by N-terminal sequencing

In addition, aKv1.1 was extracted and purified in LDAO to verify that the protein resembled its native conformation. Gel-filtration showed the structure is a tetramer.

Results: There exists a high propensity for this system to

produce IM proteins fully folded in their native conformations

Mutational Disruption of Mistic’s Structure and Function

Mutational Disruption of Mistic’s Structure and Function

• Mutations at three potentially structurally disruptive sites within the core of the protein: W13, Q36, and M75.

• Results show that Mistic’s structure is essential to its ability to chaperone cargo proteins to the bacterial lipid bilayer.

• For example:The single mutation of a core methionine (Met75) to alanine destabilized Mistic’s structure such that it partitioned between the membrane and the cytoplasm. This resulted in no protein expression when fused to aKv1.1

• Mutations at three potentially structurally disruptive sites within the core of the protein: W13, Q36, and M75.

• Results show that Mistic’s structure is essential to its ability to chaperone cargo proteins to the bacterial lipid bilayer.

• For example:The single mutation of a core methionine (Met75) to alanine destabilized Mistic’s structure such that it partitioned between the membrane and the cytoplasm. This resulted in no protein expression when fused to aKv1.1

W: Tryptophan

M: Methionine

Q: Glutamine

Conclusion:Conclusion: All available data suggest that Mistic must autonomously

associate with the bacterial membrane and that this property alone accounts for its high efficiency in chaperoning the production and integration of downstream cargo proteins.

Conformational flexibility, such as rotation of the four helices about their helical axes or even partial unraveling of the helical bundle, may allow Mistic to adapt to lipid environments.

Mistic retains an unexpectedly hydrophilic surface for an IM protein even though it is assembled internally with a typical hydrophobic core.

Mistic’s ability to help produce high yields of eukaryotic integral membrane proteins has and will enhance research in that area greatly.

All available data suggest that Mistic must autonomously associate with the bacterial membrane and that this property alone accounts for its high efficiency in chaperoning the production and integration of downstream cargo proteins.

Conformational flexibility, such as rotation of the four helices about their helical axes or even partial unraveling of the helical bundle, may allow Mistic to adapt to lipid environments.

Mistic retains an unexpectedly hydrophilic surface for an IM protein even though it is assembled internally with a typical hydrophobic core.

Mistic’s ability to help produce high yields of eukaryotic integral membrane proteins has and will enhance research in that area greatly.

ReferencesReferences

1. Roosild, Tarmo P., Jason Greenwald, Mark Vega,Samantha Castronovo, Roland Riek, and Senyon Choe. "NMR Structure of

Mistic, a Membrane-Integrating Protein for Membrane Protein Expression." Science. 25 Feb. 2005. Web. <www.sciencemag.org>.

1. Roosild, Tarmo P., Jason Greenwald, Mark Vega,Samantha Castronovo, Roland Riek, and Senyon Choe. "NMR Structure of

Mistic, a Membrane-Integrating Protein for Membrane Protein Expression." Science. 25 Feb. 2005. Web. <www.sciencemag.org>.