Protein plasticity to the extreme: changing the topology of a 4-α-helical bundle with a single amino acid substitution

Glykos, Nicholas M.; Cesareni, Gianni; Kokkinidis, Michael

doi:10.1016/s0969-2126(99)80081-1

Cited by 57 publications

(54 citation statements)

References 30 publications

(58 reference statements)

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“…58 (A backbone overlay of the 10 lowest energy NMR structures is shown.) Subsequently, this motif has been identified in a wide variety of proteins, including other dimeric helical bundles (FIS DNA-binding protein 116 and Rop mutant 68 ), intramolecularly folded helical bundles (FAS death domain 117 and N-terminal domain of the δ subunit of the ATPsynthase 118 ), and β-sheet proteins (the IgG fold 119 ), and multimeric proteins with both α/β structures (p53 tetramerization domain 120 ). Glu7 is not involved in stabilizing the native state of α 2 D. Superposition of the 10 lowest energy NMR-derived structures (left panel) shows that Glu7 adopts multiple conformations typical of solvent-exposed residues (hydrophobic core residues in gray).…”

Section: Resultsmentioning

confidence: 99%

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

et al. 2000

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De novo protein design has proven to be a powerful tool for understanding protein folding, structure, and function. In this Account, we highlight aspects of our research on the design of dimeric, four-helix bundles. Dimeric, four-helix bundles are found throughout nature, and the history of their design in our laboratory illustrates our hierarchic approach to protein design. This approach has been successfully applied to create a completely native-like protein. Structural and mutational analysis allowed us to explore the determinants of native protein structure. These determinants were then applied to the design of a dinuclear metal-binding protein that can now serve as a model for this important class of proteins.Protein folding is an important and multifaceted scientific problem. 1-8 One facet of this problem involves the prediction of three-dimensional structure from amino acid sequence, an endeavor the importance of which has been highlighted by the recent release of gene sequences of many whole organisms. As the size of the protein structural database expands, it will be increasingly possible to assign amino acid sequences to a given structural family on the basis of the homology of their sequences with proteins of known structure. Another facet of the folding problem is the delineation of the kinetic mechanism by which an unfolded protein chain undergoes the transition from a random coil with an astronomical number of configurations to a uniquely folded structure. A final facet of the protein folding problem is to understand, at the atomic level, the detailed physicochemical features that kinetically and thermodynamically direct the formation of an unique, cooperatively folded, native structure. This latter endeavor is particularly important as it lays the groundwork for the design of novel inhibitors of natural proteins as well as the construction of novel proteins and related biopolymers not found in nature.De novo protein design is an approach to the protein folding problem that critically tests our understanding of all these facets of this problem. 9,10 This method involves the design of a sequence that is intended to fold into a predetermined three-dimensional structure without the sequence being patterned after any natural protein. Through an iterative process of design and rigorous characterization, the principles governing protein folding and function can be evaluated. Thus, "failures" highlight gaps in our understanding, whereas "successes" confirm the principles used in the design and often provide simple model systems to further

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Section: Resultsmentioning

confidence: 99%

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

et al. 2000

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“…Still, Rop has afforded many surprises that could not easily be predicted, such as the topological inversion of a core variant repacked with Ala and Ile, 27 or the topological rearrangement of the A31P mutant to a ''bisecting U.'' 28 Recently, we introduced a means of measuring the melting temperatures of 96 variants of a protein at once (HighThroughput Thermal Scanning, or HTTS), and we demonstrated its use with Rop core variants. 23 However, the buried Cys residues in the core at positions 38 and 52 complicate the mutagenesis of Rop, because destabilized mutants are prone to the formation of adventitious disulfides.…”

Section: Discussionmentioning

confidence: 99%

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

et al. 2010

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Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild-type Rop is a homodimeric four-helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native state, Rop has two buried, reduced cysteine residues in its core, but these are prone to oxidation in destabilized variants, particularly upon extended storage. To circumvent this problem, we designed and characterized a Cys-free variant of Rop, including solving the 2.3 Å X-ray crystal structure. We show that the C38A C52V variant has similar structure, stability and in vivo activity to wild-type Rop, but that it has dramatically faster unfolding kinetics like virtually every other mutant of Rop that has been characterized. This cysteine-free Rop has already proven useful for studies on solution topology and on the relationship of core mutations to stability. It also suggests a general strategy for removal of pairs of Cys residues in proteins, both to make them more experimentally tractable and to improve their storage properties for therapeutic or industrial purposes.

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“…For example, Thr phosphorylation activates yeast glycogen phosphorylase by refolding the N-terminal loop into an ␣-helix (32), and several studies have identified mutations that cause changes in the topological fold of the RNA-binding protein Rop (33,34). Another example in which both backbone and side-chain conformational changes have a significant role in binding specificity is the affinity maturation of germ-line antibodies (35,36).…”

Section: Comparison With Other Mj Tyrrs Structuresmentioning

confidence: 99%

Structural plasticity of an aminoacyl-tRNA synthetase active site

Turner

Graziano

Spraggon

et al. 2006

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

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Recently, tRNA aminoacyl-tRNA synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl aminoacyl-tRNA synthetases that charge the unnatural amino acids p-bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substratebound WT synthetase, as well as a mutant synthetase that charges p-acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the ␣8-helix, which spans the WT active site. The high degree of structural plasticity that is observed in these aminoacyltRNA synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids.x-ray crystal structure ͉ unnatural amino acids ͉ expanded genetic code ͉ molecular evolution W ith the rare exceptions of selenocysteine (1) and pyrrolysine (2, 3), the common 20 amino acids are conserved across all known organisms. However, there does not appear to be an inherent limit to the size or chemical nature of the genetic code, since it has been shown that additional amino acids can be genetically encoded in both prokaryotic and eukaryotic organisms in response to nonsense or frameshift codons (4, 5). This requires a unique codon-suppressor tRNA pair and the corresponding aminoacyl-tRNA synthetase, which do not crossreact with the amino acids, tRNAs, or synthetases of the host organism (4, 5). The specificity of the aminoacyl tRNA synthetase is then altered by generating large libraries of active-site mutants and passing them through positive and negative selections to identify synthetases that selectively acylate the cognate tRNA with the unnatural amino acid but not any of the common amino acids. This approach has been used to add Ͼ30 unnatural amino acids to the genetic codes of bacteria, yeast, and mammalian cells with high fidelity and efficiencies.In Escherichia coli, a tyrosyl aminoacyl-tRNA synthetase (TyrRS) tRNA CUA Tyr pair from the archea Methanococcus jannaschii (Mj) was used as an orthogonal tRNA synthetase pair (6). Mutant synthetases have been evolved that selectively aminoacylate their cognate suppressor tRNAs with glycosylated (7), photoreactive (8), and metal-binding amino acids, as well as amino acids with unique functional groups (9-11) . To establish the molecular basis for the surprising adaptability of this synthetase, we solved (12) the structure of a mutant Mj TyrRS that is selective for p-acetylphenylalanine (p-AcPhe). The x-ray crystal structure revealed significant structural changes within the enzyme active site that result from the mutations Y32L, D158G, I159C, and L162R. The Y32L and D158G mutations remove two hydrogen bonds (H-bonds) with the...

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Protein plasticity to the extreme: changing the topology of a 4-α-helical bundle with a single amino acid substitution

Abstract: The results suggest a possible new mechanism for the creation and evolution of topological motifs, show the importance of loop regions in determining the allowable folding pathways, and illustrate the malleability of protein structures.

Cited by 57 publications

References 30 publications

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

De NovoDesign of Helical Bundles as Models for Understanding Protein Folding and Function

Cysteine‐free rop: A four‐helix bundle core mutant has wild‐type stability and structure but dramatically different unfolding kinetics

Structural plasticity of an aminoacyl-tRNA synthetase active site

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