NMR structure of an F-actin-binding “headpiece” motif from villin

Vardar, D.; Buckley, Deirdre A.; Frank, Benjamin; McKnight, C. James

doi:10.1006/jmbi.1999.3321

Cited by 70 publications

(204 citation statements)

References 34 publications

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“…The WT structure consists of a three-helix cluster surrounding a compact central core of three Phes ( Fig. 1) and differs significantly from the NMR structures of HP36 solved in isolation (Protein Data Bank ID code 1VII) (8) or within the larger headpiece (Protein Data Bank ID code 1QQV) (29). These structures were RMS-fitted by using their main-chain atoms (the most accurate part of each residue), and coordinate differences (RMSD) were calculated.…”

Section: Resultsmentioning

confidence: 92%

High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein

Chiu

Kubelka

Herbst‐Irmer

et al. 2005

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

218

290

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The 35-residue subdomain of the villin headpiece (HP35) is a small ultrafast folding protein that is being intensely studied by experiments, theory, and simulations. We have solved the x-ray structures of HP35 and its fastest folding mutant [K24 norleucine (nL)] to atomic resolution and compared their experimentally measured folding kinetics by using laser temperature jump. The structures, which are in different space groups, are almost identical to each other but differ significantly from previously solved NMR structures. Hence, the differences between the x-ray and NMR structures are probably not caused by lattice contacts or crystal͞solution differences, but reflect the higher accuracy of the x-ray structures. The x-ray structures reveal important details of packing of the hydrophobic core and some additional features, such as crosshelical H bonds. Comparison of the x-ray structures indicates that the nL substitution produces only local perturbations. Consequently, the finding that the small stabilization by the mutation is completely reflected in an increased folding rate suggests that this region of the protein is as structured in the transition state as in the folded structure. It is therefore a target for engineering to increase the folding rate of the subdomain from Ϸ0.5 s ؊1 for the nL mutant to the estimated theoretical speed limit of Ϸ3 s ؊1 .kinetics ͉ temperature jump ͉ NMR R ecent developments in both experimental and computational methods have opened exciting new possibilities for combining experiments with all-atom molecular dynamics (MD) simulations of protein folding. The dramatic improvement in time resolution in protein folding kinetic studies brought about by the introduction of nanosecond optical triggering methods (1-4), together with the use of distributed computing, now make it possible to directly compare MD simulations with experiments on ultrafast folding proteins (5). Such simulations are important because, if accurate, they contain a complete atomic description of the folding mechanism. The computational cost of folding simulations demands small size and ultrafast folding. HP35 is a subdomain of the headpiece of actinbinding protein villin and is the smallest naturally occurring polypeptide that folds autonomously without any cofactor or disulfide bond (6). Unlike other ''miniproteins'' (7), HP35 is highly thermostable (6) and, as revealed by the NMR structure, globular with a well packed hydrophobic core (8). These properties resemble much larger single-domain helical proteins and have made HP36 (HP35 plus an N-terminal Met) the focus of several all-atom folding simulations (9-17), which have thus far been based on the NMR structure.Because of the delicate balance of stabilization forces involved in protein folding an accurate structure for the folded state is essential for MD simulations of folding and protein redesign calculations. We have previously shown that HP35 is one of the fastest folding proteins, with a folding rate (Ϸ0.2 s Ϫ1 ) approaching its theoretical limit (3 s Ϫ1 ...

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Section: Resultsmentioning

confidence: 92%

High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein

Chiu

Kubelka

Herbst‐Irmer

et al. 2005

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

218

290

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“…HP36 is at the C terminus of the actin-bundling protein villin and is the C-terminal subdomain of the villin headpiece. The structure of the subdomain is essentially identical to the same region in the intact headpiece (31)(32)(33). The sequence of the protein is MLSDEDFKAVFG-MTRSAFANLPLWKQQNLKKEKGLF.…”

Section: Resultsmentioning

confidence: 95%

Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain

Brewer

Tang

et al. 2005

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

148

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Equilibrium Fourier transform infrared (FTIR) and temperaturejump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 s at 60.2°C) and double phenylalanine mutant (3.01 s at 49.9°C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 s at 49.9°C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 s compared with 6.97 s for the WT at 49.9°C.protein folding ͉ Fourier transform IR ͉ temperature jump ͉ diffusion collision T he mechanism by which an unfolded polypeptide chain folds into its final native structure is still not fully understood, despite the obvious importance of the protein folding problem. In recent years there has been considerable interest in small single-domain proteins that fold quickly. These proteins provide attractive systems for computational and theoretical studies, and they are useful experimental models of the early steps in the assembly of more complicated folds. The helical subdomain derived from the villin headpiece, HP36, is a small three-helix structure found in the extreme C terminus of villin ( Fig. 1). Its modest size and helical structure suggest that it should fold rapidly, and this hypothesis has been independently confirmed by fluorescence-detected temperature-jump (T-jump) studies and NMR lineshape analysis (1-3). The helical subdomain is probably the smallest occurring natural sequence that has been shown to fold cooperatively. Its small size and very rapid folding have made it the focus of a large number of theoretical and computational studies (4-18).Residual structure and interactions in the unfolded state may play an important role in determining the rate of protein folding. Unfolded state structure might contribute to rapid folding by constraining and limiting the initial conformational search in the unfolded basin. Some models of folding postulate an important role for residual unfolded state structure. The diffusion collision model, for example, has been applied to rapidly folding helical proteins, and a key parameter in the model is the intrinsic stability of the individual microdomains (11,19,20). Although there have been a number of experimental studies of fast folding proteins, surprisingly little work has been reported that ...

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“…In the case of thymosin ␤4, the motif is known to be important for the interaction with actin (14,15); for the villin headpiece this has been proposed (22,30). For each of the 12 libraries we obtained at least 2-5 ϫ 10 6 transformants/g DNA in E. coli, far in excess over the expected 64 variants for each position in the motif.…”

Section: Resultsmentioning

confidence: 99%

“…The proteins, thus lacking the E-tag sequence, were purified following the protocol in Ref. 22. The K d values for the villin headpiece mutants were determined in a sedimentation assay.…”

Section: Construction Of the Libraries And Rescue Of The Recombinantmentioning

confidence: 99%

“…A seemingly related sequence (LKKEKG) is present in a set of C-terminal headpiece domains (15,16) implicated in F-actin binding (20). NMR of the villin headpiece showed that the first five amino acids of the motif are in a ␣-helical conformation (21,22), and some of these are part of a charged crown suggested to be important for actin binding (20). Although both modules appear to have analogous motifs in their primary structure, they display different specificities for G-and F-actin.…”

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confidence: 99%

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A Phage Display-based Method for Determination of Relative Affinities of Mutants

Rossenu

Leyman²,

Dewitte

et al. 2003

Journal of Biological Chemistry

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We propose phage display combined with enzymelinked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein. Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate the relative affinities of a large number of mutants. As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin. The hexapeptide motifs LKKTET, present in thymosin ␤4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed. Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin. In addition, our data show that the second and the fourth amino acid of the thymosin ␤4 motif and the first three residues of the villin headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.As a consequence of various genome-sequencing projects, novel proteins are being identified, many of which may take part in new interactions. Efforts are going on to tackle the discovery of protein-protein interactions globally (1), but there is also need for novel methods for the analysis of these interactions, preferably easy, reliable, and high through-put techniques. Several means to probe amino acids that contribute to the binding of two proteins have been developed. X-ray crystallography and NMR may yield superior information about the interface of proteins at atomic resolution (2, 3). However, both methods are intrinsically difficult (even for noncomplexed proteins) and time-consuming and require expensive and sophisticated equipment, as well as large amounts of biological material. Cross-linking of interacting proteins followed by mass spectroscopy or conventional sequence determination of covalently coupled peptide fragments has limited application (4 -6). Probing the accessible surface in protein complexes by deuterium exchange has also been employed (7). But by far the two most popular methods are the use of peptide mimetics either in solution (8) or on membranes (9, 10) and especially site-directed mutagenesis (11, 12). However, in the latter powerful technique to study functions of proteins or interactions between proteins, one often faces the difficulty of choosing the position and type of amino acid exchange to be introduced. To circumvent this, one may perform a saturation mutagenesis at several positions thought to be important for the interaction. Obviously this creates a new problem, i.e. screening a large number of mutants in a systematic way.We propose to combine saturation mutagenesis, phage display and ELISA 1 for the systematic screening and quantification of pr...

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NMR structure of an F-actin-binding “headpiece” motif from villin

Cited by 70 publications

References 34 publications

High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein

High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein

Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain

A Phage Display-based Method for Determination of Relative Affinities of Mutants

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