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News: Dr. Kalodimos is the recipient of the Raymond and Beverly Sackler International Prize in the Physical Sciences

Research highlights

Molecular Chaperones - Structural basis for antifolding activity

Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.These results are now published in Nature. Read the article.


Flagellum machinery - Recognition and targeting mechanisms

The flagellum is a highly sophisticated organelle rotated by a motor that confers swarming motility to bacterial cells. Such motility is essential for the full pathogenicity of several viru- lence bacteria. Several proteins are required for the assembly and operation of the flagellum. We reported the structural characterization of FliT, a key flagellar chaperone, in the unli- ganded state and in complex with two substrate flagellar proteins. FliT adopts an autoinhibited structure in order to avoid futile interactions with the export gate in the absence of a substrate. Substrate binding to FliT activates complex tar- geting to the export gate followed by either the export of the substrate or its assembly to the export apparatus. These results are now published in Proc. Natl. Acad. Sci. USA. Read the article.


Cell invasion - Stimulated by CypA in the Abl-Crk signaling pathway

Cyclophilin A is overexpressed in a number of human cancer types, but the mechanisms by which the protein promotes oncogenic properties of cells are not understood. We now demonstrate that CypA binds the CrkII adaptor protein and prevents it from switching to the inhibited state. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly upregulated. These results are now published in Nature Chemical Biology. Read the article.


Molecular Chaperones - Structural basis for their activity

Molecular chaperones prevent aggregation and misfolding of proteins in the cellular environment and are thus central to maintaining protein homeostasis. Despite the central importance of the binding of chaperones to unfolded proteins, the structural basis of their interaction remains poorly understood. The scarcity of structural data on complexes between chaperones and unfolded proteins is primarily due to technical challenges originating in the size and dynamic nature of these complexes. We used NMR to characterize the binding of the 48 kDa unfolded alkaline phosphatase (PhoA) to the 50 kDa Trigger Factor (TF) chaperone. We obtained atomic insight into the dynamic binding and determined the solution structure of PhoA captured in an extended, unfolded state by three TF molecules (a complex of ~200 kDa).These results are now published in Science. Read the article.

Gamerdinger and Deuerling wrote a Perspective entitled "Trigger Factor Flexibility". Read the Perspective.


Protein Activity - Allosteric inhibition by suppressing excited states

The ability to inhibit binding or enzymatic activity is key to preventing aberrant behaviors of proteins. Allosteric inhibition is desirable as it offers several advantages over competitive inhibition, but the mechanisms of action remain poorly understood in most cases. We show that allosteric inhibition can be effected by destabilizing a low-populated conformational state that serves as an on-pathway intermediate for ligand binding, without altering the protein’s ground-state structure. Structure information on such important intermediates can ultimately result in more efficient design of allosteric inhibitors.These results are now published in Nature Chemical Biology. Read the article.

Gianluigi Veglia wrote a News & Views entitled "Catch them if you can". Read the News & Views. Read a technical highlight at PSI-Nature.


Bacterial Virulence - Targeting of chaperone-substrates to the ATPase

Targeting of type III secretion proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone CesAB to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB, but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon binding of its physiological substrate, EspA. These results are now published in Cell Reports. Read the article.


Protein Activity & Allostery - Regulation by conformational entropy

How the interplay between protein structure and internal dynamics regulates protein function is poorly understood. Often, ligand binding, post-translational modifications and mutations modify protein activity in a manner that is not possible to rationalize solely on the basis of structural data1. It is likely that changes in the internal motions of proteins have a major role in regulating protein activity, but the nature of their contributions remains elusive, especially in quantitative terms. Here we show that changes in conformational entropy can determine whether protein–ligand interactions will occur, even among protein complexes with identical binding interfaces. These results are now published in Nature. Read the article.

Baldwin and Kay wrote a News & Views entitled "Dynamic Binding". Read the News & Views. Read a research highlight in Nat. Struct. Mol. Biol.


Leukemogenic Activity of Bcr-Abl - Structure of its substrate CrkL

CrkL is a key adaptor signaling protein that mediates the leukemogenic activity of Bcr-Abl, but its structure has hitherto remained unknown. We have determined the solution structures of CrkL in its unphosphorylated and phosphorylated form. The data show that CrkL forms a constitutive complex with Abl thus explaining the strong preference of Bcr-Abl for CrkL. The results also highlight how the structural organization of the modular domains in adaptor proteins can control signaling outcome. These results are now published in Nature Chemical Biology. Read the article.

Kobashigawa and Inagaki wrote a News & Views entitled "CrkL is not Crk-like". Read the News & Views.


Protein-Protein Interactions - Regulation by structural instability

We have discovered a novel regulatory mechanism in protein-protein interactions mediated by finely-tuned structural instability coupled with molecular mimicry. We have solved the structure of a chaperone adopting a molten globule conformation and found that packing defects allow transient exposure of the substrate binding site. Correction of the structural instability in the chaperone results in a non-functional system. The substrate binds to the chaperone using molecular mimicry to counteract autoinhibition. These results are now published in Molecular Cell. Read the article.


Protein Activity - Regulation by a proline switch

We have determined the structural basis for the regulation of the Crk proto-oncogenic protein by a proline switch. Proline isomerization toggles Crk between two conformations: an autoinhibitory, stabilized by the cis form, and an activated conformation promoted by the trans form. In addition to acting as a structural switch the heterogeneous proline recruits cyclophilin A, which accelerates the interconversion rate between the isomers thereby regulating the kinetics of Crk activation. The data provide atomic insight into the mechanisms that underpin the functionality of this binary switch. These results are now published in Nature Chemical Biology. Read the article. Watch the movie.

Nicholson and De wrote a News & Views entitled "The twist in Crk signaling revealed". Read the News & Views.


Protein Function - Allostery goes dynamic

We have discovered a very intriguing mechanism that calls for revision of our fundamental understanding of the mechanisms that underlie protein function and regulation. Our study suggests that the notion of purely structurally regulated activity in allosteric proteins should be revised to include a frequently dominating contribution from protein dynamics. We have characterized cAMP binding to CAP. We find, surprisingly, that even when in a structurally inactive conformation, CAP can be activated for ligand (DNA) binding by changes in protein dynamics. These results are now published in Nature. Read the Editor's summary. Read the article. F1000 evaluation.


Gene Regulation - Structural basis for cAMP-mediated activation of CAP

We have determined the solution structure of the catabolite activator protein (CAP) in the absence of cAMP. The structure, together with previously determined structures of CAP in the presence of cAMP and DNA, provide the complete structural basis for cAMP-mediated allosteric activation of CAP. These results are now published in Proc. Natl. Acad. Sci. USA. Read the article. Watch the movie.

Ma and Nussinov wrote a commentary in PNAS entitled “Amplification of signaling via cellular allosteric relay and protein disorder”. Read the commentary. Read a research highlight. F1000 evaluation.


Protein Secretion - Structural basis for signal sequence recognition

Recognition of signal sequences by cognate receptors is a decisive step in correctly sorting secretory from nonsecretory proteins. We have determined the solution structure of a secretion signal sequence in complex with an essential bacterial receptor, SecA, the 204-kDa ATPase motor of the Sec translocase machinery. The combined data not only explain how SecA may achieve the promiscuous recognition of a large set of signal sequences, but also provide insight into how the Sec nanomachinery may ultimately be assembled. These results are now published in Cell. Read the article. F1000 evaluation.


Signal Transduction - Control by proline isomerization

We have shown that autoinhibition can be controlled by an intrinsic intramolecular switch afforded by prolyl cis-trans isomerization. Peptidyl-prolyl isomerase enzymes such as cyclophilin A accelerate the slow cis-trans intervonversion rate. Proline isomerization appears to make an ideal switch that can regulate the kinetics of activation, thereby modulating the dynamics of signal response. These results are now published in Molecular Cell. Read the article

Nicholson & Ping Lu wrote a Preview entitled "Prolyl cis-trans isomerization as a molecular timer in Crk signaling". Read the Preview. F1000 evaluation.


Protein Allostery - It can be dynamically mediated

We have shown that allosteric interactions can be mediated exclusively by transmitted changes in protein motions, in the absence of conformational changes. Our results provide strong support of the existence of purely dynamics-driven allostery. These data are now published in Nature Structural & Molecular Biology. Read the article

Lewis Kay discussed our paper in the "Journal Club" of Nature; "The molecular dance of a protein allows a chemist's secret wish to come true". Read the highlight


Protein Secretion - SecA helicase motor properties

SecA is an exquisitely designed and engineered protein motor that recognizes secretory proteins and couples their transport through the transmembrane SecYEG channel to ATP binding and hydrolysis. Using an integrated NMR, thermodynamic, and biochemical approach we have discovered a novel mechanism that underlies its function. The results are published in Nature Structural & Molecular Biology (article of the month). Download the article

John Hunt and coworkers wrote a News & Views entitled "Disorder breathes life into a DEAD motor". Read the News & Views


Transcription Regulation - Structural basis

Sequence-specific protein-DNA interactions are responsible for the regulation of key biological functions such as replication of the genome, initiation of transcription, and repair of damaged DNA. We have recently provided the structural and dynamic basis of the 
complete recognition pathway of the lac repressor system. The results are published in Science. Read the article

Peter von Hippel wrote a nice Perspective entitled "Completing the view of transcriptional regulation". Read the Perspective. F1000 evaluation.

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