The N-terminal to C-terminal motif in protein folding and function

Mallela, Krishna; Englander, S. Walter

doi:10.1073/pnas.0409114102

Cited by 144 publications

(115 citation statements)

References 84 publications

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“…These peptides represent protein segments that are sequentially distant but are adjacent in the native structure (Fig. 4 B-D) where they come together to close the longest siteto-site loop in the native protein, analogous to some previous results (33). They form the two helices and two of the four β-strands pictured in Fig.…”

Section: Significancementioning

confidence: 73%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

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

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107

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Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.SAXS | HDX | protein collapse | denatured state ensemble F ifty years after Anfinsen's seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3-7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8-12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work...

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Section: Significancementioning

confidence: 73%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

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

Self Cite

107

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“…5) and that isomerization of the Vik1 neck may be the actuator that "flips" a molecular switch controlling Vik1's release from the microtubule to allow motility. As noted by Khalil et al (4), most kinesins, and approximately half of all single-domain proteins in the Protein Data Bank (43), have interacting N-and C-terminal elements that experience dynamic interconversion between different types of secondary structure, or undergo disorder-to-order transitions. It appears that Vik1 may have adapted this behavior for regulation of its microtubule binding interactions by Kar3 in lieu of nucleotide binding and enzymatic function.…”

Section: Discussionmentioning

confidence: 99%

Neck Rotation and Neck Mimic Docking in the Noncatalytic Kar3-associated Protein Vik1

Duan

Jia

Joshi

et al. 2012

Journal of Biological Chemistry

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Background: Kar3Vik1 is a heterodimeric kinesin with one catalytic subunit (Kar3) and one noncatalytic subunit (Vik1). Results: Vik1 experiences conformational changes in regions analogous to the force-producing elements in catalytic kinesins. Conclusion: A molecular mechanism by which Kar3 could trigger Vik1's release from microtubules was revealed. Significance: These findings will serve as the prototype for understanding the motile mechanism of kinesin-14 motors in general.It is widely accepted that movement of kinesin motor proteins is accomplished by coupling ATP binding, hydrolysis, and product release to conformational changes in the microtubule-binding and force-generating elements of their motor domain. Therefore, understanding how the Saccharomyces cerevisiae proteins Cik1 and Vik1 are able to function as direct participants in movement of Kar3Cik1 and Kar3Vik1 kinesin complexes presents an interesting challenge given that their motor homology domain (MHD) cannot bind ATP. Our crystal structures of the Vik1 ortholog from Candida glabrata may provide insight into this mechanism by showing that its neck and neck mimic-like element can adopt several different conformations reminiscent of those observed in catalytic kinesins. We found that when the neck is ␣-helical and interacting with the MHD core, the C terminus of CgVik1 docks onto the central ␤-sheet similarly to the ATP-bound form of Ncd. Alternatively, when neck-core interactions are broken, the C terminus is disordered. Mutations designed to impair neck rotation, or some of the neck-MHD interactions, decreased microtubule gliding velocity and steady state ATPase rate of CgKar3Vik1 complexes significantly. These results strongly suggest that neck rotation and neck mimic docking in Vik1 and Cik1 may be a structural mechanism for communication with Kar3.Eukaryotic cells rely on nanometer-sized motors called kinesins to transport cellular components along microtubules (1) or to help build the mitotic spindle and distribute chromosomes between daughter cells (2,3). Recent studies have shown that dynamic interactions between the neck and a short region of either the N or C terminus of the motor domain form a structure responsible for force generation by the neck (4 -7) and that its conformation and interactions with the motor domain core, or regulatory proteins, is linked to the nucleotide-and microtubule-binding state of the motor (8, 9). In kinesin-1, this region forms an N-terminal extension of the motor domain, called the "cover strand" (5), and in kinesin-14 motors this region is at the C terminus, after the ␣6 helix, and has been dubbed the "neck mimic" (8).Kar3 is a kinesin-14 that plays essential roles in mitosis, meiosis, and karyogamy in Saccharomyces cerevisiae and Candida albicans (10 -13). These include cross-linking, stabilizing, and sliding spindle pole microtubules, as well as depolymerizing microtubules (10,14). To accomplish this array of functions, Kar3 associates with two discrete regulatory subunits, Cik1 and Vik1 (14 -16), whose mot...

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“…According to the "foldons" theory [43], the 50-54 helix is the segment identified as the one unfolding during the first denaturation transition. The C-terminal helix (residues 88-102) is close to the mutated region; however, the region constituted by the C-terminal and the N-terminal helices is very stable, being the first region to fold and the last to unfold in the folding-unfolding process of cyt c [43,44].…”

Section: Discussionmentioning

confidence: 99%

The key role played by charge in the interaction of cytochrome c with cardiolipin

et al. 2016

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mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys → Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein.Abstract Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c-CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such Electronic supplementary material The online version of this article

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The N-terminal to C-terminal motif in protein folding and function

Cited by 144 publications

References 84 publications

Folding of a large protein at high structural resolution

Folding of a large protein at high structural resolution

Neck Rotation and Neck Mimic Docking in the Noncatalytic Kar3-associated Protein Vik1

The key role played by charge in the interaction of cytochrome c with cardiolipin

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