Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system

Kükenshöner, Tim; Wohlwend, Daniel; Niemöller, Christoph; Dondapati, Padmarupa; Speck, Janina; Adeniran, Adebola; Nieth, Anita; Gerhardt, S.; Einsle, Oliver; Müller, Kristian M.; Arndt, Katja M.

doi:10.1016/j.jsb.2014.03.002

Cited by 11 publications

(8 citation statements)

References 60 publications

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“…It should be noted though that the prediction of oligomerization preference is associated with errors. While cases discussed above represent naturally evolved interfaces, the iM10 binding to the microphthalmia associated transcription factor, MITF ( 4c7n), was optimized by directed evolution in bacterial, in vivo selection system . This selection process results in similar fitness pressures compared to the natural systems, as it occurs in the crowded environment of the cell, where factors as oligomerization, affinity, and specificity may come into play.…”

Section: Discussionmentioning

confidence: 99%

Computational assessment of folding energy landscapes in heterodimeric coiled coils

André

Bjelic

2018

Proteins

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The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs-into-holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.

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Section: Discussionmentioning

confidence: 99%

Computational assessment of folding energy landscapes in heterodimeric coiled coils

André

Bjelic

2018

Proteins

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“…The path length of the cuvette was 2 mm. Similarly to our previous study, CD spectra were recorded between 300 and 180 nm at −8, 5, and 20 °C, before and after the thermal denaturation run to confirm reversibility of the unfolding process. For each spectrum, three scans were averaged.…”

Section: Materials and Methodsmentioning

confidence: 96%

“…L-Peptides were designed and synthesized with an N-cap (Ala-Ser) and a C-cap (Gly-Ala-Pro) sequence to satisfy unpaired backbone hydrogen bonds at both helix ends and acetylated N- and amidated C-termini (Protein Peptide Research, Wickham, U.K.). Purification was performed via reverse-phase HPLC using an acetonitrile gradient, as previously described . D-Acid was synthesized with an N-terminal biotin substituent and a Gly-Lys-Gly-linker (Lys = L-amino acid) to provide a protease cleavage site for phage elution.…”

Section: Materials and Methodsmentioning

confidence: 99%

Analysis of Selected and Designed Chimeric D- and L-α-Helix Assemblies

Kükenshöner

Hagemann²,

Wohlwend³

et al. 2014

Biomacromolecules

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D-peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

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“…In order to distinguish whether the altered interaction behavior of the proline substitution mutants was indeed related to the helix breaking properties of proline or if the substitution sites simply constitute crucial interaction sites, we additionally substituted all investigated amino acid positions by alanine (L27A, I45A, L65A and I69A). Similar to leucine and isoleucine, alanine has a high helical propensity and would thus not be expected to interfere with helix formation (Kukenshoner et al, 2014;Mason et al, 2009;Moitra et al, 1997). However, due to its small side chain, substitutions to alanine would abrogate specific residue-residue interactions at the respective sites (Cunningham and Wells, 1989;Morrison and Weiss, 2001).…”

Section: Helix Breaking Point Mutations Impair the Interaction Abilitmentioning

confidence: 99%

Structural requirements of the phytoplasma effector protein SAP54 for causing homeotic transformation of floral organs

Aurin¹,

Haupt

Goerlach

et al. 2019

Preprint

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SIGNIFICANCE STATEMENTPhytoplasmas are bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins such as SAP54, which leads to the degradation of some floral homeotic proteins. Our study clarifies the structural requirements of SAP54 function and illuminates the molecular mode of interaction and thus may ultimately help to develop treatments against some devastating plant diseases. SummaryPhytoplasmas are intracellular bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins. One of these effector proteins, termed SECRETED ASTER YELLOWS-WITCHES' BROOM PROTEIN 54 (SAP54), leads to the degradation of a specific subset of floral homeotic proteins of the MIKC-type MADSdomain family via the ubiquitin-proteasome pathway. In consequence, the developing flowers show the homeotic transformation of floral organs into vegetative leaf-like structures. The molecular mechanism of SAP54 action involves physical binding to the keratin-like K-domain of MIKC-type proteins, and to some RAD23 proteins, which translocate ubiquitylated substrates to the proteasome. The structural requirements and specificity of SAP54 function are poorly understood, however. Here we report, based on biophysical and molecular biological analyses, that SAP54 folds into α-helical structures. We also show that the insertion of helixbreaking mutations disrupts correct folding of SAP54, which interferes with the ability of SAP54 to bind to its target proteins and to cause disease phenotypes in vivo. Surprisingly, dynamic light scattering data together with electrophoretic mobility shift assays suggest that SAP54 preferentially binds to multimeric complexes of MIKC-type proteins rather than to dimers or monomers of these proteins. Together with literature data this finding suggests that MIKC-type proteins and SAP54 constitute multimeric α-helical coiled-coils, possibly also involving other partners such as RAD23 proteins. Our investigations clarify the structurefunction relationship of an important phytoplasma effector protein and thus may ultimately help to develop treatments against some devastating plant diseases.

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Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system

Cited by 11 publications

References 60 publications

Computational assessment of folding energy landscapes in heterodimeric coiled coils

Computational assessment of folding energy landscapes in heterodimeric coiled coils

Analysis of Selected and Designed Chimeric D- and L-α-Helix Assemblies

Structural requirements of the phytoplasma effector protein SAP54 for causing homeotic transformation of floral organs

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