PAS (PER-ARNT-SIM) domains are a family of sensor protein domains involved in signal transduction in a wide range of organisms. Recent structural studies have revealed that these domains contain a structurally conserved α/β-fold, whereas almost no conservation is observed at the amino acid sequence level. The photoactive yellow protein, a bacterial light sensor, has been proposed as the PAS structural prototype yet contains an N-terminal helix-turn-helix motif not found in other PAS domains. Here we describe the atomic resolution structure of a photoactive yellow protein deletion mutant lacking this motif, revealing that the PAS domain is indeed able to fold independently and is not affected by the removal of these residues. Computer simulations of currently known PAS domain structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins.

# MATERIALS AND METHODS

## Crystallization, Diffraction, and Refinement

Δ25PYP encompassing residues 26–125 of PYP was expressed and purified as described previously (32). Crystals were grown by equilibration of 1 μl of 30 mg/ml protein with 1 μl of mother liquor (1.8 m ammonium sulfate, 10 mmCoCl2, 100 mm MES, pH 6.5) against a 1-ml reservoir of mother liquor. Crystals appeared after 2–3 days with a largest dimension of 0.4 mm.

Diffraction data were collected at beamline ID14-EH1 (European Synchrotron Radiation Facility, Grenoble, France) and processed with the HKL package (Table I
                        ) (36). The structure of Δ25PYP was solved by molecular replacement with AMoRe (37) using the native PYP structure (Protein Data Bank code 2PHY) (7) as a search model (excluding the chromophore) against 8–4-Å data. A solution was found (r = 0.479, correlation coefficient = 0.282) with two molecules in the asymmetric unit. Initial refinement was carried out with CNS (38) interspersed with model building in O (39). The chromophore was not included in the refinement until it was well defined by an unbiased F
                        
                           o
                         − F
                        
                           c
                         ,φcalc map (Fig. 1
                        ). Further rounds of refinement with SHELX97 (40) allowed the placement of water molecules and the assignment of some alternate side chain conformations. In the last stages of the refinement, hydrogen atoms were included (Table I).

Residues 113 (leucine) and 114 (serine) in one monomer and residue 116 (aspartic acid) in the other monomer were disordered, although some evidence for several possible conformations was visible in the map. The building of these regions was attempted, but their conformations could not be determined with confidence. Similar observations were made in previous crystallographic studies of PYP in the P65 space group (30, 31, 41). At the N terminus, the well defined electron density was present for Ala-27 at the early stages of refinement. Subsequent maps also defined the conformation of Leu-26.

## Computational Details

### CONCOORD Simulations

Sampling of conformational space by the computer simulation method CONCOORD (42) was performed for crystal structures of the PAS domains depicted in Fig. 2
                              . Besides these existing PAS domain structures, the Δ25PYP crystal structure described here was also simulated. As a negative control for the subsequent comparisons, a CONCOORD ensemble starting from the crystal structure of turkey lysozyme (Protein Data Bank code135L) bearing no structural resemblance to PAS domains was also calculated. During the CONCOORD runs, 1000 structures were generated and a damping factor of 0.25 was applied to avoid unreasonable side chain geometries.

### Essential Dynamics

Essential dynamics (43) determines concerted motions of atoms from an ensemble of structures, for example, a set of crystal structures (44-47) or a trajectory from a computer simulation (43, 48-51). Here the CONCOORD ensembles were used as input. A covariance matrix is constructed that describes the correlation of the positional shifts of one atom with those of another atom as shown in Equation 1,

Equation 1
                                    
                                       
                                          C
                                          
                                             ij
                                          
                                       
                                       =
                                       (
                                       (
                                       
                                          x
                                          
                                             i
                                          
                                       
                                       −
                                       
                                          x
                                          
                                             i
                                             ,
                                             0
                                          
                                       
                                       )
                                       (
                                       
                                          x
                                          
                                             j
                                          
                                       
                                       −
                                       
                                          x
                                          
                                             j
                                             ,
                                             0
                                          
                                       
                                       )
                                       )
                                    
                                 
                              where x
                              
                                 i
                               and x
                              
                                 j
                               represent the coordinates of atoms i and j in a conformation, whereas x
                              
                                 i
                              
                              ,0 andx
                              
                                 j
                              
                              ,0 represent the average coordinates of the atoms over the ensemble. The average is calculated over all structures after they are superimposed on a reference structure to remove overall translational and rotational motion. Diagonalizing this matrix yields a set of eigenvectors and eigenvalues. The eigenvectors are directions in a 3N-dimensional space (where Nis the number of atoms), and motion along a single eigenvector corresponds to concerted displacements of groups of atoms in Cartesian space. The eigenvalues are a measure of the mean square fluctuation of the system along the corresponding eigenvectors. The eigenvectors are sorted according to their eigenvalue, the first eigenvector having the largest eigenvalue.

To allow direct comparison of concerted motions for different proteins, an equal number of atoms must be used in the essential dynamics calculations. A first simplification is that only Cαatoms are taken into account, which sufficiently represent the large motions of the protein backbone (48, 52). When the structures also contain insertions and deletions such as in the PAS domains (Fig. 3
                              ), further simplifications will need to be applied to reduce all of the structures to a common core (44). Residues in the PAS domains that overlapped structurally were selected by the DALI server (53), which performs a pairwise comparison of secondary structure elements. The results of the pairwise alignment on secondary structure were compared to yield the common structural elements present in the PAS domains (Fig. 3). For lysozyme, the negative control, an equal number of residues was selected starting from the N terminus.

# RESULTS AND DISCUSSION

### Atomic Resolution Crystal Structure of Δ25PYP

The structure of Δ25PYP was solved by molecular replacement and refined to a 1.14-Å resolution (R-factor = 0.147,R
                              free = 0.177) (Fig. 1 and TableI). The asymmetric unit contains two protein molecules related by a non-crystallographic 2-fold rotation axis (Fig. 1). The molecules have a similar conformation with a root mean square deviation of 0.77 Å on Cα atoms. Compared with the wtPYP structure, the two molecules superimpose with root mean square deviations of 0.99 and 0.76 Å, respectively. From these superpositions, positional shifts of the Cα atoms of the mutant structures with respect to the positions of the Cαatoms in wild type PYP are given in Fig.2. The N terminus and the loops consisting of residues 84–88, 98–101, and 111–117 in Δ25PYP have a different conformation than those in wild type PYP. The different conformation of the Δ25PYP N terminus compared with the equivalent residues in wtPYP is most probably caused by the deletion of the first 25 residues (Fig.3). When the first two residues at the N terminus of Δ25PYP are excluded from the superposition, the root mean square deviation is reduced by ∼0.2 Å. From the NMR structure and the comparison of two crystal forms of wild type PYP, the loop around residue Met-100 is observed to be flexible (7, 30, 54). Close contacts between the two monomers in the asymmetric unit cell affect the conformation of the “100 loop” (Fig. 1). The distance between the backbone atoms of the two Met-100 residues is <4.0 Å and could influence the conformation of this loop. The large differences in the orientation of the 111–117 loop, which is disordered in this loop and previously reported PYP structures (30, 31, 41), and the 84–88 loop can again be explained by crystals contacts (Fig. 1).

Native PYP contains two hydrophobic cores, one within the PAS domain between the β-sheet and the αC helix and another between the β-sheet and the two small helices of the N-terminal domain (7). For the latter domain, residues Phe-28 (at the start of βA), Trp-199, and Phe-121 (both on βE) extend toward the N-terminal domain and have become solvent exposed in Δ25PYP yet appear not to self-dimerize through crystal contacts. This finding agrees with the observation that in Δ25PYP the fluorescence emission of Trp-119 (the only tryptophan in PYP) is enhanced and blue-shifted, suggesting a more polar environment (32). It is likely that it is these solvent-exposed hydrophobic residues that cause the reported decrease in temperature stability observed for Δ25PYP (32). In summary, the structural data show that the removal of the first 25 residues does not significantly affect the overall fold of the PAS domain core. This is in agreement with the spectrophotometric data on Δ25PYP, which show that the Δ25PYP absorbance maximum associated with the chemical environment of the chromophore and very sensitive to perturbation is only minimally blue-shifted (32). Similar photocycle intermediates as in wtPYP are present. Only the kinetics of the recovery reaction in the photocycle is slowed down, which again could be associated with a less stable ground state structure due to exposure of several hydrophobic residues.

As observed earlier (4, 34), none of the other currently known PAS domain structures (Fig. 3) possesses extra residues similar to the N terminus of wtPYP. Thus, this may be a unique feature that plays a specific but, as yet, unidentified role in PYP function. Although the PAS domains share a common fold, only few residues are conserved at the sequence level (Figs. 3 and 4
                              ). When these residues (mostly leucines, isoleucines, and valines) are compared among the different PAS domains, they appear to form part of a conserved hydrophobic core (Figs. 3 and 4). Interestingly, most of these residues are located in a cluster near helix αA, which appears to act as a lid on the ligand binding pockets and undergoes conformational changes in PYP (9, 11, 23, 25, 29, 30).

### PAS Domain Flexibility

Because PAS domains function as sensor proteins in signaling pathways and share a common fold, it is possible that they also have common dynamic properties, which would allow them to communicate with transducer proteins through a conserved mechanism. We have attempted to investigate this through computer simulation of all of the structurally characterized PAS domains, namely PYP (7), FixL (35), HERG (33), and LOV (34). The complete proteins were subjected to CONCOORD simulations (42) followed by extraction of the common Cα atoms as defined by a structure-based sequence alignment (Figs. 3 and 4). This encompassed 78 residues (marked in Figs. 3 and 4) including most of the central β-sheet, the αA/B helices, and part of the long αC helix. The resulting ensembles of structures were analyzed by essential dynamics (43), yielding separate sets of eigenvectors that describe concerted fluctuations of atoms for each protein. These are sorted by their corresponding eigenvalues,i.e. the first eigenvector being the one with the largest eigenvalue, revealing in all cases that the majority (>95%) of the motion is covered by the first 5% (12) of the eigenvectors. With this condensed description of flexibility in the individual PAS domains, comparisons are facilitated.

Sets of eigenvectors can be projected onto each other yielding a cumulative square inner product, indicating the degree of similarity of the motions described by the eigenvectors. Here we have focused on the first 12 eigenvectors (5% 3N = 234 total eigenvectors), because these together describe approximately 95% of the total motion in the ensembles. TableII
                               shows that the eigenvectors from the different PAS domains are very similar, suggesting that the cores of the PAS domains share common motions, which are not present in lysozyme (the negative control). This is further confirmed by projection of the PAS domain eigenvectors onto the first three eigenvectors calculated from the wtPYP ensemble (Fig. 5
                              ). Whereas the other PAS domains reproduce these largest wtPYP motions for up to 90% within the first 12 eigenvectors, they are almost absent in the lysozyme ensemble. Thus, the PAS domains not only share a common structure but also share a common conformational flexibility.

To understand the motions described by the eigenvectors on a molecular level, the minimum and maximum projections onto an eigenvector can be translated back to Cartesian space and compared as Cαtraces. In Fig. 6
                              , the minimum and maximum projections of the first 3 eigenvectors of Δ25PYP are compared. The central β-sheet appears to be relatively static, whereas the loops, most notably the αA/αB segment, show the largest fluctuations. In the PAS domains, this segment is generally important for the binding of the ligand (7, 34, 35). For instance, in PYP, residue Arg-52 on this segment is known to undergo a conformational change (9, 11, 29, 30) upon isomerization of the chromophore. Glu-46, which shares a proton with the chromophore, is also located in this region (Fig. 4). Similarly, the αA/αB segment is involved in binding the heme in FixL (35) and the FMN in LOV (34) both via interaction with a phenylalanine, which lies at the equivalent position of Glu-46 in PYP. In addition, a recent analysis of LOV domains has revealed that the αA/αB region participates in a conserved salt bridge, which is also observed in FixL and HERG and has been proposed to be involved in signal transduction (55). It is noteworthy that despite these similar interactions and conservation of conformational flexibility, there is almost no sequence conservation in the αA/αB segment.

# CONCLUSION

The data presented here show that in the absence of the N-terminal domain, PYP maintains its PAS-fold despite the exposure of several hydrophobic residues to solvent. The Δ25PYP structure together with the recently determined of the LOV domain in complex with FMN (34) allowed further structural comparisons of the PAS family. Although these proteins have almost entirely dissimilar sequences, their structures are remarkably similar with the conserved parts, the β-sheet and the αA/B helices, making up the PAS core. This finding suggests that although these proteins bind different ligands, their signaling states are reached through similar conformational changes. We investigated this by simulating the complete PAS domain proteins that have been structurally defined to date and extracting from that the structurally conserved core. An analysis of the data shows that in particular the αA/B segment moves in a concerted fashion. Thus, we propose that despite the absence of any sequence conservation, the PAS domains are not only structurally conserved but also share a common conformational flexibility that may have evolved to (i) accommodate the various input signals from different ligands/co-factors located at different positions in the domain and (ii) transmit the sensing event to downstream transducer proteins.

We thank Raafat Hamze for contributions in the early stages of the project. We thank the European Synchrotron Radiation Facility (Grenoble, France) for the time at beamline ID14-EH1.