RESUMO
FKBP12, a small human enzyme, aids protein folding by catalyzing cis-trans isomerization of peptidyl-prolyl bonds, and is involved in cell signaling pathways, calcium regulation, and the immune response. The underlying molecular mechanisms are not fully understood, but it is well-known that aromatic residues in the active site and neighboring loops are important for substrate binding and catalysis. Here we report micro- to millisecond exchange dynamics of aromatic side chains in the active site region of ligand-free FKBP12, involving a minor state population of 0.5% and an exchange rate of 3600 s-1, similar to previous results for the backbone and methyl-bearing side chains. The exchange process involves tautomerization of H87. In the major state H87 is highly flexible and occupies the common HNε2 tautomer, while in the minor state it occupies the rare HNδ1 tautomer, which typically requires stabilization by specific interactions, such as hydrogen bonds. This finding suggests that the exchange process is coupled to a rearrangement of the hydrogen bond network around H87. Upon addition of the active-site inhibitor FK506 the exchange of all aromatic residues is quenched, with exception of H87. The H87 resonances are broadened beyond detection, suggesting that interconversion between tautomers prevail in the FK506-bound state. While key active-site residues undergo conformational exchange in the apo state, the exchange rate is considerably faster than the catalytic turnover, as determined herein by Michaelis-Menten type analysis of NMR line shapes and chemical shifts. We discuss alternative interpretations of this observation in terms of FKBP12 function.
Assuntos
Aminoácidos Aromáticos/química , Domínio Catalítico , Conformação Proteica , Proteína 1A de Ligação a Tacrolimo/química , Aminoácidos Aromáticos/metabolismo , Sítios de Ligação/genética , Histidina/química , Histidina/metabolismo , Humanos , Ligação de Hidrogênio , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Ligação Proteica , Tacrolimo/química , Tacrolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid p-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein.
Assuntos
Aminoácidos/química , Corantes Fluorescentes/química , Proteínas/química , Cinética , Espectrometria de FluorescênciaRESUMO
c-CrkII is a central signal adapter protein. A domain opening/closing reaction between its N- and C-terminal Src homology 3 domains (SH3N and SH3C, respectively) controls signal propagation from upstream tyrosine kinases to downstream targets. In chicken but not in human c-CrkII, opening/closing is coupled with cis/trans isomerization at Pro-238 in SH3C. Here, we used advanced double-mixing experiments and kinetic simulations to uncover dynamic domain interactions in c-CrkII and to elucidate how they are linked with cis/trans isomerization and how this regulates substrate binding to SH3N. Pro-238 trans â cis isomerization is not a simple on/off switch but converts chicken c-CrkII from a high affinity to a low affinity form. We present a double-box model that describes c-CrkII as an allosteric system consisting of an open, high affinity R state and a closed, low affinity T state. Coupling of the T-R transition with an intrinsically slow prolyl isomerization provides c-CrkII with a kinetic memory and possibly functions as a molecular attenuator during signal transduction.
Assuntos
Proteínas Proto-Oncogênicas c-crk/química , Proteínas Proto-Oncogênicas c-crk/metabolismo , Regulação Alostérica , Animais , Galinhas , Humanos , Dobramento de Proteína , Transdução de Sinais , Domínios de Homologia de src/fisiologiaRESUMO
Secretion of proteins into the membrane-cell wall space is essential for cell wall biosynthesis and pathogenicity in Gram-positive bacteria. Folding and maturation of many secreted proteins depend on a single extracellular foldase, the PrsA protein. PrsA is a 30-kDa protein, lipid anchored to the outer leaflet of the cell membrane. The crystal structure of Bacillus subtilis PrsA reveals a central catalytic parvulin-type prolyl isomerase domain, which is inserted into a larger composite NC domain formed by the N- and C-terminal regions. This domain architecture resembles, despite a lack of sequence conservation, both trigger factor, a ribosome-binding bacterial chaperone, and SurA, a periplasmic chaperone in Gram-negative bacteria. Two main structural differences are observed in that the N-terminal arm of PrsA is substantially shortened relative to the trigger factor and SurA and in that PrsA is found to dimerize in a unique fashion via its NC domain. Dimerization leads to a large, bowl-shaped crevice, which might be involved in vivo in protecting substrate proteins from aggregation. NMR experiments reveal a direct, dynamic interaction of both the parvulin and the NC domain with secretion propeptides, which have been implicated in substrate targeting to PrsA.
Assuntos
Proteínas de Bactérias/química , Lipoproteínas/química , Proteínas de Membrana/química , Multimerização Proteica , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação ProteicaRESUMO
BACKGROUND: Prolyl cis/trans isomerizations have long been known as critical and rate-limiting steps in protein folding. RESULTS: Now it is clear that they are also used as slow conformational switches and molecular timers in the regulation of protein activity. Here we describe several such proline switches and how they are regulated. CONCLUSIONS AND GENERAL SIGNIFICANCE: Prolyl isomerizations can function as attenuators and provide allosteric systems with a molecular memory. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.
Assuntos
Prolina/química , Dobramento de Proteína , Proteínas/química , Regulação Alostérica/fisiologia , Animais , Humanos , Prolina/metabolismo , Estrutura Terciária de Proteína , Proteínas/metabolismoRESUMO
Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.
Assuntos
Proteínas e Peptídeos de Choque Frio/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Pseudoalteromonas/metabolismo , Thermotoga maritima/metabolismo , Aclimatação , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Dobramento de Proteína , Ribossomos/metabolismo , TemperaturaRESUMO
Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators.
Assuntos
Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio de Domínios Poros em Tandem/genética , Fosfolipídeos , Potenciais da Membrana , Potássio/metabolismoRESUMO
The plasma membrane protects the interiors of cells from their surroundings and also plays a critical role in communication, sensing, and nutrient import. As a result, the cell membrane and its constituents are among the most important drug targets. Studying the cell membrane and the processes it facilitates is therefore crucial, but it is a highly complex environment that is difficult to access experimentally. Various model membrane systems have been developed to provide an environment in which membrane proteins can be studied in isolation. Among them, tethered bilayer lipid membranes (tBLMs) are a promising model system providing a solvent-free membrane environment which can be prepared by self-assembly, is resistant to mechanical disturbances and has a high electrical resistance. tBLMs are therefore uniquely suitable to study ion channels and charge transport processes. However, ion channels are often large, complex, multimeric structures and their function requires a particular lipid environment. In this paper, we show that SthK, a bacterial cyclic nucleotide gated (CNG) ion channel that is strongly dependent on the surrounding lipid composition, functions normally when embedded into a sparsely tethered lipid bilayer. As SthK has been very well characterized in terms of structure and function, it is well-suited to demonstrate the utility of tethered membrane systems. A model membrane system suitable for studying CNG ion channels would be useful, as this type of ion channel performs a wide range of physiological functions in bacteria, plants, and mammals and is therefore of fundamental scientific interest as well as being highly relevant to medicine.
Assuntos
Canais Iônicos , Técnicas Eletroquímicas , Canais Iônicos/química , Bicamadas Lipídicas/química , Microscopia de Força Atômica , AMP Cíclico/metabolismo , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
Cyclic nucleotide-gated ion channels are crucial in many physiological processes such as vision and pacemaking in the heart. SthK is a prokaryotic homolog with high sequence and structure similarities to hyperpolarization-activated and cyclic nucleotide-modulated and cyclic nucleotide-gated channels, especially at the level of the cyclic nucleotide binding domains (CNBDs). Functional measurements showed that cyclic adenosine monophosphate (cAMP) is a channel activator while cyclic guanosine monophosphate (cGMP) barely leads to pore opening. Here, using atomic force microscopy single-molecule force spectroscopy and force probe molecular dynamics simulations, we unravel quantitatively and at the atomic level how CNBDs discriminate between cyclic nucleotides. We find that cAMP binds to the SthK CNBD slightly stronger than cGMP and accesses a deep-bound state that a cGMP-bound CNBD cannot reach. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos , Nucleotídeos Cíclicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Ativação do Canal Iônico/fisiologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismoRESUMO
Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos , Nucleotídeos Cíclicos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação do Canal Iônico , AMP Cíclico/metabolismo , LigantesRESUMO
Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.
Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Lipídeos de Membrana , ÂnionsRESUMO
Prolyl isomerases catalyze the cis/trans isomerization of peptide bonds preceding proline. Previously, we had determined the specificity toward the residue before the proline for cyclophilin-, FKBP-, and parvulin-type prolyl isomerases by using proline-containing oligopeptides and refolding proteins as model substrates. Here, we report the specificities of members of these three prolyl isomerase families for the residue following the proline, again in short peptide and in refolding protein chains. Human cyclophilin 18 and parvulin 10 from Escherichia coli show high activity, but low specificity, with respect to the residue following the proline. Human FKBP12 prefers hydrophobic residues at this position in the peptide assays and shows a very low activity in the protein folding assays. This activity was strongly improved, and the sequence specificity was virtually eliminated after the insertion of a chaperone domain into the prolyl isomerase domain of human FKBP12.
Assuntos
Peptidilprolil Isomerase/metabolismo , Prolina/metabolismo , Humanos , Modelos MolecularesRESUMO
Recent developments in cryogenic electron microscopy (cryo-EM) led to an exponential increase in high-resolution structures of membrane proteins, and in particular ion channels. However, structures alone can only provide limited information about the workings of these proteins. In order to understand ion channel function and regulation in molecular detail, the obtained structural data need to be correlated to functional states of the same protein. Here, we describe several techniques that can be employed to study ion channel structure and function in vitro and under defined, similar conditions. Lipid nanodiscs provide a native-like environment for membrane proteins and have become a valuable tool in membrane protein structural biology and biophysics. Combined with liposome-based flux assays for the kinetic analysis of ion channel activity as well as electrophysiological recordings, researchers now have access to an array of experimental techniques allowing for detailed structure-function correlations using purified components. Two examples are presented where we put emphasis on the lipid environment and time-resolved techniques together with mutations and protein engineering to interpret structural data obtained from single particle cryo-EM on cyclic nucleotide-gated or Ca2+-gated K+ channels. Furthermore, we provide short protocols for all the assays used in our work so that others can adapt these techniques to their experimental needs. Comprehensive structure-function correlations are essential in order to pharmacologically target channelopathies.
Assuntos
Canais Iônicos , Proteínas de Membrana , Microscopia Crioeletrônica , Cinética , Lipídeos , Proteínas de Membrana/genéticaRESUMO
SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Spirochaeta/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Ciclosporina/farmacologia , Ativação do Canal Iônico/fisiologia , Isomerismo , Cinética , Modelos Moleculares , Peptidilprolil Isomerase/metabolismo , Prolina/metabolismoRESUMO
Integral membrane proteins have historically been challenging targets for biophysical research due to their low solubility in aqueous solution. Their importance for chemical and electrical signaling between cells, however, makes them fascinating targets for investigators interested in the regulation of cellular and physiological processes. Since membrane proteins shunt the barrier imposed by the cell membrane, they also serve as entry points for drugs, adding pharmaceutical research and development to the interests. In recent years, detailed understanding of membrane protein function has significantly increased due to high-resolution structural information obtained from single-particle cryo-EM, X-ray crystallography, and NMR. In order to further advance our mechanistic understanding on membrane proteins as well as foster drug development, it is crucial to generate more biophysical and functional data on these proteins under defined conditions. To that end, different techniques have been developed to stabilize integral membrane proteins in native-like environments that allow both structural and biophysical investigations-amphipols, lipid bicelles, and lipid nanodiscs. In this chapter, we provide detailed protocols for the reconstitution of membrane proteins according to these three techniques. We also outline some of the possible applications of each technique and discuss their advantages and possible caveats.
Assuntos
Biofísica/métodos , Bicamadas Lipídicas/química , Microdomínios da Membrana , Proteínas de Membrana/química , Renaturação Proteica , Química Analítica , Detergentes/química , Detergentes/farmacologia , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Micelas , Modelos Moleculares , Nanoestruturas/química , Polímeros/química , Polímeros/farmacologia , Propilaminas/química , Propilaminas/farmacologia , Conformação Proteica , Dobramento de Proteína , Renaturação Proteica/efeitos dos fármacos , Estabilidade Proteica , SolubilidadeRESUMO
The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by electrophysiological techniques from protein heterologously expressed in Xenopus oocytes and mammalian cells. On the other hand, expression and purification of these proteins for biophysical and structural analyses in vitro is problematic and expensive and, accordingly, only limited information on the purified channels is available in the literature. Here we describe a protocol for binding studies of fluorescently labeled cyclic nucleotides to a homologue of eukaryotic CNG channels. Furthermore, we describe how to directly probe binding of unlabeled cyclic nucleotides in a competition assay. The use of fluorescence as a sensitive probe for ligand binding reduces the amount of protein needed and enables fast and easy measurements using standard laboratory equipment.
RESUMO
Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide-modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.
Assuntos
Proteínas de Bactérias/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação do Canal Iônico , Animais , Proteínas de Bactérias/química , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Bicamadas Lipídicas/metabolismo , Ligação Proteica , Spirochaeta , XenopusRESUMO
Cyclophilins are ubiquitous cis-trans-prolyl isomerases (PPIases) found in all kingdoms of life. Here, we identify a novel family of cyclophilins, termed AquaCyps, which specifically occurs in marine Alphaproteobacteria, but not in related terrestric species. In addition to a canonical PPIase domain, AquaCyps contain large extensions and insertions. The crystal structures of two representatives from Hirschia baltica, AquaCyp293 and AquaCyp300, reveal the formation of a compact domain, the NIC domain, by the N- and C-terminal extensions together with a central insertion. The NIC domain adopts a novel mixed alpha-helical, beta-sheet fold that is linked to the cyclophilin domain via a conserved disulfide bond. In its overall fold, AquaCyp293 resembles AquaCyp300, but the two proteins utilize distinct sets of active site residues, consistent with differences in their PPIase catalytic properties. While AquaCyp293 is a highly active general PPIase, AquaCyp300 is specific for hydrophobic substrate peptides and exhibits lower overall activity.
Assuntos
Alphaproteobacteria/enzimologia , Proteínas de Bactérias/química , Ciclofilinas/química , Catálise , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Prolyl isomerizations are intrinsically slow processes. They determine the rates of many protein folding reactions and control regulatory events in folded proteins. Prolyl isomerases are able to catalyze these isomerizations, and thus, they have the potential to assist protein folding and to modulate protein function. Here, we provide examples for how prolyl isomerizations limit protein folding and are accelerated by prolyl isomerases and how native-state prolyl isomerizations regulate protein functions. The roles of prolines in protein folding and protein function are closely interrelated because both of them depend on the coupling between cis/trans isomerization and conformational changes that can involve extended regions of a protein.
Assuntos
Peptidilprolil Isomerase/metabolismo , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas/fisiologia , Animais , Catálise , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismoRESUMO
The signal adapter protein c-CrkII from chicken but not from human uses isomerization at Pro238 in the SH3C domain to regulate the activity of the SH3N domain. The different behavior of human and chicken c-CrkII originates from only two differences in sequence, at positions 239 after Pro238 and 272 in the N-Src loop of SH3C. We analyzed the kinetics of substrate binding to SH3N and an assay for its coupling with Pro238 isomerization in SH3C to identify the molecular path from Pro238 to the substrate binding site of SH3N. The transâcis isomerization at Pro238 and a relocation of Phe239 re-organize the energetics of a hydrophobic cluster in the N-Src loop of SH3C and re-shape this region to optimize its interactions with SH3N. Concomitantly, the backbone becomes strained at Met272. We suggest that, in human c-CrkII, movement at position 239 and strain at position 272 are not tolerated because the ß-branched residues Ile239 and Val272 restrain the backbone mobility and thus destabilize the cis Pro238 form.