Packing of apolar side chains enables accurate design of highly stable membrane proteins

Mravic, Marco; Thomaston, Jessica L.; Tucker, Maxwell R.; Solomon, Paige; Liu, Lijun; DeGrado, William F.

doi:10.1126/science.aav7541

Cited by 102 publications

(102 citation statements)

References 77 publications

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“…This indicates a strict steric requirement for van der Waals packing interactions between TM domain side chains that dictates the stability and regulation of the αvβ3 resting state. Similar Ile-Leu mutations have been observed to abrogate select TM domain interactions for shorter engineered TM peptides (34,35), yet it is noteworthy to find a similar result within a large protein complex interacting simultaneously through several extramembrane domains. The mutational consequences might also be rationalized in the context of previous reports for small-x 3 -small motifs.…”

Section: Discussionmentioning

confidence: 54%

Unique transmembrane domain interactions differentially modulate integrin αvβ3 and αIIbβ3 function

Litvinov

Mravic

Zhu

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Lateral transmembrane (TM) helix–helix interactions between single-span membrane proteins play an important role in the assembly and signaling of many cell-surface receptors. Often, these helices contain two highly conserved yet distinct interaction motifs, arranged such that the motifs cannot be engaged simultaneously. However, there is sparse experimental evidence that dual-engagement mechanisms play a role in biological signaling. Here, we investigate the function of the two conserved interaction motifs in the TM domain of the integrin β3-subunit. The first motif uses reciprocating “large-large-small” amino acid packing to mediate the interaction of the β3 and αIIb TM domains and maintain the inactive resting conformation of the platelet integrin αIIbβ3. The second motif, S-x3-A-x3-I, is a variant of the classical “G-x3-G” motif. Using site-directed mutagenesis, optical trap-based force spectroscopy, and molecular modeling, we show that S-x3-A-x3-I does not engage αIIb but rather mediates the interaction of the β3 TM domain with the TM domain of the αv-subunit of the integrin αvβ3. Like αIIbβ3, αvβ3 on circulating platelets is inactive, and in the absence of platelet stimulation is unable to interact with components of the subendothelial matrix. However, disrupting any residue in the β3 S-x3-A-x3-I motif by site-directed mutations is sufficient to induce αvβ3 binding to the αvβ3 ligand osteopontin and to the monoclonal antibody WOW-1. Thus, the β3-integrin TM domain is able to engage in two mutually exclusive interactions that produce alternate α-subunit pairing, creating two integrins with distinct biological functions.

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Section: Discussionmentioning

confidence: 54%

Unique transmembrane domain interactions differentially modulate integrin αvβ3 and αIIbβ3 function

Litvinov

Mravic

Zhu

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Notably, previous experiments which truncated the polar upper-TM region of an engineered, watersoluble form of PLB found that this also served as a switch from pentamer to tetramer, which could suggest a greater role for polar forces in higher-order PLB oligomerization (Slovic, Lear et al 2005). However, emerging research has also continued to demonstrate the prominent role of van der Waal forces in stabilizing peptide oligomers and an engineered PLB-like molecule devoid of strongly polar residues has even been shown to easily stabilize as a pentamer (Mravic, Thomaston et al 2019), which we suggest is secondary to hydrophobic zipper interactions. The evidence presented here show PLB and SLN as examples supporting the prominent role of hydrophobic interactions in small peptide oligomerization as well as determination of oligomer order.…”

Section: Novel Structural Insights Into Sln and Plb Self-assemblymentioning

confidence: 99%

Phospholamban and sarcolipin share similar transmembrane zipper motifs that control self-association affinity and oligomer stoichiometry

DesLauriers

Svensson

Thomas

et al. 2019

Preprint

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We have characterized the structural determinants of phospholamban (PLB) and sarcolipin (SLN) self-association using site-directed mutagenesis, SDS-PAGE, and fluorescence resonance energy transfer (FRET) microscopy. PLB and SLN are single-pass transmembrane (TM) peptides that are critically involved in regulation of contractility in cardiac and skeletal muscle via reversible inhibition of calcium (Ca) transport by SERCA. PLB and SLN also exhibit ion channel activity in vitro, yet the physiological significance of these functions is unknown. Here we have determined that structural insights offered by the tetrameric PLB Cys41 to Leu (C41L) mutation, a mutant with four possible leucine/isoleucine zipper interactions for stabilizing PLB tetramers. Using scanning alanine mutagenesis and SDS-PAGE, we have determined the C41L-PLB tetramer is destabilized by mutation of Leu37 to Ala (L37A) or Ile40 to Ala (I40A), which are the same a-and d-arm residues stabilizing the PLB pentamer via leucine/isoleucine zippers, highlighting the importance of these two zippers in PLB higher-order oligomerization. The new possible zipper arm in C41L-PLB (N34, C41L, I48) did not contribute to tetramerization. On the other hand, we determined that tetramer conversion back to pentamer was induced by alanine mutation of Ile48, a residue located on the e-arm below C41L, implicating steric interaction and restriction are the stabilizing and destabilizing forces that control the distribution between pentamer and tetramer populations. We propose that the e-arm and hydrophobic residues in the adjacent b-arm act as secondary structural motifs that help control the stoichiometry of PLB oligomerization. FRET microscopy and alanine mutagenesis of SLN residues Val14 (V14A) or Leu21 (L21A) decreased the binding affinity of the SLN-SLN complex, demonstrating the importance of each residue in mediating self-association. Helical wheel analysis supports a heptad-repeat TM zipper mechanism of SLN oligomerization, similar to the 3.5 residue/turn Leu and Ile zippers found in PLB pentamers. Collectively, our studies add new insights on the conservation of homologous hydrophobic 3-4 pattern of residues in zipper motifs that mediate PLB and SLN self-assembly. We propose that the importance of these apolar, steric interactions in the TM domain are widespread in stabilizing higher-order oligomerization of membrane proteins.Structural determinants of SLN and PLB self-association 2 1 Abbreviations and Acronyms: Amp, ampicillin; Ca, calcium; CAT; chloramphenicol acetyltransferase; CFP, cyan fluorescent protein; Cl -, chloride; DOPC, dioleoylphosphatidylcholine; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; his-CAT, chloramphenicol transferase with a 6-residue histidine tag fused to the N-terminus; his-PLB, phospholamban with a 6-residue histidine tag fused to the N-terminus; his-SLN, sarcolipin with a 6-residue histidine tag fused to the N-terminus; IPTG, isopropyl β-D-1-

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“…Automating modelling and design processes and extending them to complex membrane proteins will likely require an accurate energy function that correctly balances intra-protein interactions, membrane solvation and water solvation 13,14 .…”

Section: Introductionmentioning

confidence: 99%

A lipophilicity-based energy function for membrane-protein modelling and design

Weinstein

Elazar

Fleishman

2019

Preprint

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Membrane-protein design is an exciting and increasingly successful research area which has led to landmarks including the design of stable and accurate membrane-integral proteins based on coiled-coil motifs. Design of topologically more complex proteins, such as most receptors, channels, and transporters, however, demands an energy function that balances contributions from intra-protein contacts and protein-membrane interactions. Recent advances in water-soluble all-atom energy functions have increased the accuracy in structure-prediction benchmarks. The plasma membrane, however, imposes different physical constraints on protein solvation. To understand these constraints, we recently developed a high-throughput experimental screen, called dsTβL, and inferred apparent insertion energies for each amino acid at dozens of positions across the bacterial plasma membrane. Here, we express these profiles as lipophilicity energy terms in Rosetta and demonstrate that the new energy function outperforms previous ones in modelling and design benchmarks. Rosetta ab initio simulations starting from an extended chain recapitulate two-thirds of the experimentally determined structures of membrane-spanning homo-oligomers with <2.5Å root-mean-square deviation within the top-predicted five models. Furthermore, in two sequence-design benchmarks, the energy function improves discrimination of stabilizing point mutations and recapitulates natural membrane-protein sequences of known structure, thereby recommending this new energy function for membrane-protein modelling and design.

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Packing of apolar side chains enables accurate design of highly stable membrane proteins

Cited by 102 publications

References 77 publications

Unique transmembrane domain interactions differentially modulate integrin αvβ3 and αIIbβ3 function

Unique transmembrane domain interactions differentially modulate integrin αvβ3 and αIIbβ3 function

Phospholamban and sarcolipin share similar transmembrane zipper motifs that control self-association affinity and oligomer stoichiometry

A lipophilicity-based energy function for membrane-protein modelling and design

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