Estructura y distancias entre poblaciones

CypA-catalysed prolyl isomerisation can have a profound impact on many key proteins in diverse

cellular processing, including cell growth, stress responses, cell signalling, immune response, viral

infection, cytokinesis. Unlike covalent modification or global unfolding, proline isomerisation is an

intrinsic conformational exchange process that has the potential to direct ligand recognition and

to control protein activity w ithin the confines o f the native state (Andreotti 2003). Indeed, o f the

naturally occurring aa, proline is unique and appears to fulfil multiple roles in the context o f

native, folded proteins. In some proteins, proline isomerisation may confer conformer-specific

properties to a native protein fold by modulating the features o f a protein surface. Alternatively, a

conformational heterogeneous proline residue may cause minimal structural perturbations, but

could instead serve as a recognition site for PPIase. The c/s and trans isomers o f many proteins

have distinct functions, and their conversions by PPIases can function as a new general class o f

protein regulatory switches. Indeed, the cis and trans isomers provide stable local motifs that

differ dramatically in structure, thereby providing a mechanism fo r selecting distinct pools of

binding partners, even in the context o f otherwise unstructured regions o f proteins. Therefore,

cis-trans isomerisation is a molecular switch that can be used in an enzyme-regulated manner to

help control the tim ing of biological events such as isomer-specific protein-protein interactions, as

well as the amplitude and duration of a cellular process (Lu 2007).

NMR and X-ray studies have shown CypA in complex with the HIV-1 capsid protein (Gamble 1996;

Campos-Olivas 1999) and w ith the interleukin-2 tyrosine kinase (Itk) (Brazin 2002; Andreotti

2003). In these cases a similar equilibrium o f the cis-trans isomerisation state ratio o f the

substrates has been observed, that is altered when they are in complex w ith CypA, suggesting

that an increase in the cis conformation o f the bound substrate may be a general mechanism for

CypA to mediate the control o f the protein function.

The HIV-1 capsid protein forms the conical core structure at the center o f the mature virion and

can bind w ith high affinity human CypA, thereby packaging it into the virion. Through this

interaction CypA is able to promote both the formation and the infectivity o f human

immunodeficiency virus (Luban 1993; Bosco 2002; Braaten 1996 and 2001; Sokolskaja 2004). The

N-terminal portion o f HIV-1 capsid protein binds efficiently human CypA's active site (Howard

2003; Gamble 1996; Campos-Olivas 1999) (Figure 17); in particular, CypA binds to an exposed

proline-rich loop in the HIV-1 capsid protein A (CA) domain o f HIV-1 Gag envelope protein that has

been demonstrated to have 44,4% identity w ith the sequence o f another CypA interactor, that is

CD147 (Yurchenko 2002). This indicates that microorganisms can also use prolyl cis-trans

isomerisation as a regulatory mechanism to gain entry into host cells. The binding site fo r CypA, 102

Gly89-Pro90 in capsid, exists in the cis and trans forms and CypA can catalyse Gly89-Pro90

isomerisation, thus suggesting a role in maturation or disassembly o f viruses. Indeed, conditions

that disrupt CA-CypA interaction, such as mutational inactivation o f the CypA binding site or

inhibition by CsA, inhibit replication o f most HIV-1 strains, and HIV-1 replication is attenuated in a

human T-cell line that lacks CypA (Braaten 1996). In this case, the Cyp-CA interaction seems to

block the recognition o f the HIV-1 virion by the host cell restriction factor R efl, an intrinsic

antiviral cellular protection mechanism (Howard 2003; Towers 2003). CypA was also shown to

enahance HCV RNA replication stimulating the RNA binding ability o f the NS5A protein,

modulating the conformation o f the RNA binding m o tif by cis-trans isomerisation (Foster 2011).

Figure 17 CypA/HIV-1 capsid complex.

X-ray structure of the N-terminal portion of the HIV-1 capsid (yellow) interacting with the PPIase cavity of CypA (green). The CypA residues involved in the catalytic actvity are shown in blue. A loop of the capsid protein (that contains 4 prolines) can undergo the cis-trans isomerisation and the following conformational switch (Howard 2003).

In addition, CypA may also have an im portant role in the immune response through the

interaction w ith the tyrosine kinase Itk, a non-receptor protein that participates in the

intracellular signaling events leading to Th2 cell activation. NMR structural studies combined w ith

mutational analysis have shown that CypA can bind to the Itk SH2 domain which is generally in a

cis conformation, and catalyse the cis-trans isomerisation o f the Asn286/Pro-287 imide bond (Figure 18). The cis conformer o f Itk is likely an inactive form o f the enzyme and CypA acts as a

repressor, thus maintaining its inactivated form. CypA PPIase activity, via interaction w ith Itk and

proline-dependent conformational switch, can thus regulate substrate recognition and mediate

regulatory interactions in CD4+T-cell activation (Brazin 2002; Colgan 2004; Andreotti 2003).

Plasma Membrane and trans conformers. The self-associated, dimeric form of Itk predominates for the cis imide bond containing conformer (left), while the trans imide bond within the SH2 domain (denoted SH2*) favours interactions with signaling partners, such as Slp-76 (right). Interconversion between the two conformations of Itk likely occurs via the monomeric form of the enzyme (center). Itk catalytic activity is inhibited by the PPIase activity o f CypA. Adapted from Colgan 2004.

The importance o f CypA PPIase activity in cell signalling has also been documented. Crk is a

member o f an adaptor protein fam ily that has several SH2 and SH3 domains. NMR studies have

revealed that cis-trans isomerisation o f the prolyl bond at position Gly237-Pro238 (located

between tw o o f the SH3 domains) is very slow and that PPIase binding to the SH3 domain

facilitates an opening o f the "gate," where the isomerisation rate is increased and the adaptor

forms an "open," less restricted active conformation, that is catalysed by CypA (Figure 19) (Sarkar

2007).

104

cis

Figure 19 Model of the equilibrium of conformational states of Crk.

The intramolecular inhibitory SH3N/SH3C interaction is stabilised by the c/s conformer of the Gly237- Pro238 prolyl bond, whereas the trans conformer favours an uninhibited state. Activation occurs by PPII ligand binding to a low population of uninhibited states wherein SH3N binding site is accessible, thereby shifting the equilibrium towards the SH3N-PPII ligand bound state. Pro238 acts as a molecular switch that has the intrinsic capacity to regulate the autoinhibition of Crk. Adapted from Sarkar 2007.

CypA constitutively associates w ith prolactin receptor (PRLr) and Janus kinase 2 (Jak2) (Syed 2003)

(Figure 20), resulting in enhanced Jak2 and Stat5 activity. In particular, CypA directly acts on PRLr

through Pro-334, mediating a conformational change within the PRLr/Jak2 complex. Thus, CypA is

required fo r effective PRLr-induced signaling, a mechanism particularly relevant in the progression

o f human breast cancer (Zheng 2008).

(i) CypA is constitutively associated with PRLr and Jak2 during unstimulated conditions, (ii) Upon binding of PRL to PRLr, CypA positively regulates Jak2 activity by exerting its isomerase activity, (iii) presumably through its switching function of a cis-trans peptidyl prolyl isomerase. Ablation of CypA PPI activity by cyclosporine A (CsA) and other approaches inhibits PRL signaling and might be a novel therapeutic strategy in the treatment of human breast cancer. The red arrow in (ii) denotes cis-trans interconversion of proline 334 in the X-box m otif of the PRLr; the red circle containing P indicates phosphorylation of JAK2 kinase, PRLr, and signal transducer and activator of transcription 5a (Stat5a). Adapted from Clevanger 2009.

These findings suggest that prolyl isomerisation is emerging as a complementary regulatory

mechanism to control signalling networks.