Ribosome-associated factor Y adopts a fold resembling a double-stranded RNA binding domain scaffold Keqiong Ye 1 , Alexander Serganov 1 , Weidong Hu 1 , Maria Garber 2 and Dinshaw J. Patel 1 1 Cellular Biochemistry & Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, USA; 2 Institute of Protein Research, Moscow Region, Pushchino, Russia Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits trans- lation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a babbba topology and represents a compact two- layered sandwich of two nearly parallel a helices packed against the same side of a four-stranded b sheet. The 23 C-terminal residues of the protein are disordered. Long- range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C-terminal region of helix 1 and the loop linking this helix with strand b2 show significant conformational exchange in the ms–lstime scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both a helices, which most probably constitutes an RNA binding site. The observed babbba topology of pY resembles the abbba topology of double-stranded RNA-binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs. Keywords: backbone dynamics; dsRBD; molecular align- ment; residual dipolar couplings; RNA-binding domain. Protein expression is finely tuned in the cell allowing for adaptations to various environmental changes. The regula- tion strategies adopted by the cell encompass almost every aspect of the protein production process, including mRNA transcription, translation in the ribosome and protein degradation. Ribosome activity constitutes an important target in the control of protein expression. Protein Y, the product of yfiA gene, is a ribosome-associated protein present in E. coli and many other bacteria [1–3]. pY was earlier assigned to the r 54 modulation protein family based on the observation that mutations in the related down- stream r 54 gene cause an increase in the level of expression from r 54 -dependent promoters [4]. pY (also called ribosome associated inhibitor, RaiA) [2] was detected in the ribosome fraction during environmental stress, as a consequence of either low temperature [2] or excessive cell density [2,3]. Furthermore, pY binds to the small ribosomal subunit in an Mg 2+ -dependent manner and becomes less exposed tosolvent upon association of the small and large ribosomal subunits. This suggests an intersubunit position for pY in the 70S ribosome and may explain its ribosome stabilization effect [1]. pY inhibits translation most probably by interfering with the binding of the aminoacyl- tRNA to the ribosomal A site [2]. It has been proposed that A-site blocking by pY arrests protein synthesis during cold shock or at the stationary phase of cell culture, thereby mediating overall adaptation of bacteria to environmental stress. pY shows about 40% identity with the protein encoded by E. coli yhbH gene. The associationof pY and 70S ribosomes contrasts with the preferential binding of YhbH and the dimerized 100S ribosomes in the stationary phase of cell growth [3]. The authors suggested that these related proteins play a stabilization role during ribosome storage. The protein Y is widely dispersed in bacteria. Homologs of pY are also found in two plants, Arabidopsis thaliana and Spinacia oleracea. Interestingly, the nuclear encoded distinct homolog from spinach (formerly known as CS-5, PSrp-1, S22, or S30 according to the nomenclature of Schmidt et al. [5]) was found in the stroma of chloroplasts as well as in the small ribosomal subunits [6,7]. In spite of the larger size and relatively low homology with its E. coli counterpart, the ribosome binding site of S30 most probably resembles the binding site of pY because the S30 protein can be incorpor- ated into E. coli ribosomes in vivo [8]. Another pY homolog, protein LtrA from cyanobacteria Synechococcus PCC 7002, is expressed only in the dark [9], suggesting that members of the pY family can generally be involved in the process of adaptation to the environmental conditions. Correspondence to D. J. Patel, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. Fax: + 1212 7173066, Tel.: + 1212 6397207, E-mail: pateld@mskcc.org Abbreviations: dsRBD, double-stranded RNA binding domain; HSQC, heteronuclear single quantum coherence; P, protection factor; pY, protein Y; R1, longitudinal relaxation rate; R2, transversal relaxation rate; RDC, residual dipolar coupling; r.m.s.d., root mean square deviation. Note: The PDB ID code for the pY structures is 1L4S. The access number of NMR assignments at the Biomolecular Magnetic Resonance Databank is 5315. (Received 20 May 2002, revised 22 August 2002, accepted 30 August 2002) Eur. J. Biochem. 269, 5182–5191 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03222.x In the present study, we have determined the solution structure of pY from E. coli by NMR spectroscopy using NOE and residual dipolar coupling restraints. The protein has a babbba arrangement of secondary elements and a disordered 23-residue C-terminal tail. An analysis of the surface conserved residues in the pY structure suggest a potential RNA-binding site residing within the C-termini of two a helices. A comparison with the recently published similar structure of the protein HI0257 from Haemophilus influenzae is also presented [10]. MATERIALS AND METHODS Sample preparation The gene encoding 112 residues of pY was amplified by PCR with the genomic E. coli DNA as the template, and after purification and digestion by restriction enzymes, was subcloned into NdeIandEcoRV sites of the pET29b vector (Novagen). The protein was overproduced in E. coli BL21(DE3) strain. Uniformly 15 N-enriched protein was prepared by growing cells in M9 minimal medium contain- ing 15 NH 4 Cl. Uniformly 15 N, 13 C-labeled protein was puri- fied from cells grown in a 1 : 1 mixture of Celton CN medium (Martek Biosciences) and M9 medium with 15 NH 4 Cl and 2 gÆL )1 [ 13 C]glucose. To obtain 10% 13 C- labeled protein, cells were grown in the M9 medium containing 10% of [ 13 C]glucose. All minimal media were supplemented with basal medium eagle vitamin solution (Life Technologies Gibco BRL) and trace element supple- ments [11]. The purification procedure was as follows: cells producing pY were disrupted by sonication in the buffer containing 20 m M Tris/HCl, pH 7.0, 50 m M NaCl, 2 m M MgCl 2 , and protease inhibitors. Cell debris was removed by low-speed centrifugation (30 000 g) for 30 min, and ribo- somes were precipitated by ultra-speed centrifugation (150 000 g) for 3 h. The pH of the supernatant was adjusted to 5.5 with 1.0 M sodium acetate, pH 5.0. The precipitate was removed by low-speed centrifugation, and the super- natant was loaded onto an SP Sepharose (Amersham Pharmacia Biotech Inc.) column equilibrated with the buffer containing 20 m M sodium acetate (pH 5.5) and 50 m M NaCl. The protein was eluted using an NaCl gradient. The protein containing fractions were combined, concentrated in an Amicon cell, and diluted 10 fold with 10 m M Tris/HCl, pH 9.0. The protein was loaded onto a MonoQ column (Amersham Pharmacia Biotech Inc.) equilibrated with buffer containing 50 m M Tris/HCl (pH 9.0) and 50 m M NaCl, and eluted using the NaCl gradient. Finally, the protein was purified by gel-filtration on a HiLoad 16/60 Superdex 75 column (Amersham Pharmacia Biotech Inc.) in buffer containing 10 m M sodium phosphate (pH 5.7) and 50 m M NaCl. Standard NMR samples were prepared containing approximately 1–2 m M pY protein in gel-filtration buffer supplemented with 0.3 m M NaN 3 in either 93% H 2 O/7% 2 H 2 O or 100% 2 H 2 O. NMR spectroscopy NMR spectra were recorded at 31.5 °ConaVarianUnity plus 600 MHz spectrometer equipped with a triple-reson- ance probe and Z-axis gradient. Spectra were processed using the FELIX 98 (MSI Inc.) software and analyzed using NMRVIEW 5.0 software [12]. The 1 H, 15 N, 13 C¢,and 13 C a backbone resonances were assigned using triple resonance CBCA(CO)NH, HNCACB, HNCO, and HN(CA)CO experiments collected on a 15 N, 13 C-labeled sample [13]. The side-chain resonances were assigned from H(CCO)NH, C(CO)NH, HCCH-TOCSY ( 2 H 2 O), 15 N-TOCSY and 2D-TOCSY ( 2 H 2 O) experiments. The b-methylene stereospecific assignments were obtained by a qualitative comparison of intensities in the 3D HNHB, HACAHB-COSY, 15 N-NOESY and 13 C-NOESY spectra. v1 torsion angle restraints (including Ile, Thr and Val) were introduced in the form of the common staggered confor- mations (v1 ¼ ) 60, 60, 180°), with an error of ±20 ° .All methyl groups in Leu and Val residue were stereospecifically assigned using a 10% 13 C-labeled sample [14]. Qualitative 3 J HNHa coupling constants were measured from an HNHA [15] experiment using a correction factor of 0.9 to compen- sate the different relaxation properties of the diagonal and cross peaks. 3 J HNHa values were directly used in structural refinement [16]. Additionally, dihedral angle constraints of )65(±25) ° and )120(±30) ° for / were set for 3 J HNHa <5.8 Hz and >8.0 Hz, respectively. Distance restraints were determined using NOEs derived from the 3D 15 N-NOESY (100 ms), 3D 13 C-NOESY ( 2 H 2 O, 80 ms), and from the aromatic region in a 2D NOESY ( 2 H 2 O, 140 ms). Upper bounds of distance con- straints were classified into the ranges of 2.7, 3.3, 4.0, 5.0, and 6.0 A ˚ based upon relative NOE volumes of the cross peaks. The lower bounds were set to 0.0 A ˚ in order to use ambiguous NOEs in the calculation [17]. Molecular alignment with bacteriophage Pf1 was not successful because of protein association with phage [18]. Weak alignment of the protein (7.3 Hz splitting of the deuterium signal) was finally obtained in a 3.2% (w/v) bicelle [19] solution in 20 m M potassium phosphate buffer (pH 6.6) and 1 m M NaN 3 at 40 °C. Bicelles were prepared by mixing dimyristoylglycerophosphocholine, dihexanoyl- phosphatidylcholine, and cetyltrimethylammonium bro- mide at a molar ratio of 30 : 10 : 2. Premixed dimyristoylglycerophosphocholine/dihexanoylphosphatidyl choline was purchased from Avanti. Cetyltrimethylammo- nium bromide was added to stabilize bicelles [20]. The neutral pH required for bicelle stability affected the positions of some peaks in the 1 H- 15 N- heteronuclear single quantum coherence (HSQC) spectrum, although peaks in 1 H- 13 C-HSQC were much less shifted. Only peaks that could be reliably tracked were analyzed. One-bond coupling constants for 1 J N-NH and 1 J Ca-Ha were measured using a J-modulated intensity based method [21]. Estimated errors were less than 1 Hz. Isotropic data were recorded under standard conditions. Residual dipolar couplings were determined as the difference between coupling constants in the aligned and isotropic conditions, and were incorpor- ated at the late stage of structure calculation. One-bond 1 D CH values were converted to equivalent 1 D NH values. Data for residues with 1 H- 15 N heteronuclear NOE values less than 0.6 were excluded from the structure calculations. The axial component (D a ) and rhombicity (R)ofthe alignment tensor were estimated from the powder pattern of the residual dipolar coupling histogram [22], and from singular value decomposition analysis of the well defined secondary structures [20]. The values were then optimized Ó FEBS 2002 Solution structure of ribosome associated factor Y (Eur. J. Biochem. 269) 5183 using a grid search to minimize the energy of the lowest energy structure. Small deviations from the optimal align- ment tensor had no significant effects on the total energy and calculated structures. The final values of D a and R were 19 Hz and 0.3, respectively. The bond length between pseudo-tetraatoms representing the principle order system in the structure calculation was modified from the default value of 1 A ˚ to 10 A ˚ . Such modification resulted in lower overall CNS energy and increased convergence rate. Hydrogen exchange rates were measured from a series of 1 H- 15 N-HSQC spectra recorded at 31.5 °Cafterthe addition of lyophilized protein in 2 H 2 O. The spectra were recorded at 11, 19, 27, 35, 43, 51, 59, 67 and 75 min. The exchange rate k ex was obtained by fitting the intensity decay to a single exponential function. The protection factor P was calculated as k rc /k ex ,wherek rc is the exchange rate for amino acid in random coil conforma- tion [23]. Potential hydrogen bond constraints set to each observed proton in the first recorded spectrum were used in the later stages of calculation, when a unique hydrogen bond acceptor could be identified from the structure model. Every hydrogen bond constraint was represented by two distance constraints of 1.7–2.3 A ˚ (between H and O) and 2.7–3.3 A ˚ (between N and O) across the N–HO hydrogen bond alignment. 15 N relaxation parameters (R1, R2 and NOE) were measured by standard methods [24]. The R1 relaxation times were set to 5.4, 54.1, 162.4, 270.6, 378.8, 487.1, 595.3, 703.6, 811.8, 920.0, 1028.3 and 1136.5 ms. The R2 relaxa- tion time was set to 8, 16, 32, 48, 64, 80, 96, 112, 128, 144, 160 and 176 ms. The peak intensities from different relaxation times were fit to a single exponential curve using the program GNUPLOT (ftp://ftp.darmouth.edu/pub/gnu- plot). Errors were reported by the fitting program. 1 H- 15 N steady-state NOE values were obtained by recording spectra with and without 1 H saturation of 3-s duration, and by calculating the ratios of the peak intensities. The NOE errors were estimated by the root-mean-square value of the background noise. Structure calculations Structures were calculated using the simulated annealing method with torsion angle dynamics implemented in CNS 1.0 program [25]. Employed energy functions were: quadratic harmonic potential for covalent geometry, soft square-well quadratic potentials for the experimental distance and torsion angle restraints, harmonic potentials for the 3 J HNHA coupling constants and dipolar coupling constants, and a quadratic van der Waals repulsion term for the nonbonded contacts. Ambiguous NOEs were assigned by NMRVIEW in an ARIA-like manner [17] and were used throughout the structure calculations. Final NOEs constraints were assigned by NMRVIEW with a Nilges cut-off value 0.85, and by manual inspection. Constraints for nonstereospecifically assigned prochiral protons were set to the loosest values as described by Fletcher et al. [26]. Only constraints from well-ordered residues (1–90) were used in structure calculation. Figures were prepared using MOLMOL [27] and GRASP [28]. RESULTS AND DISCUSSION Assignments Mass spectrometry analysis of the unlabeled pY revealed that the N-terminal methionine was completely removed from the protein, which was overproduced in E. coli cells using the T7 phage RNA polymerase-based overexpression system [29]. The 1 H- 15 N-HSQC spectrum of uniformly labeled pY (Fig. 1) shows all the features of a well-folded, conformationally homogeneous protein, although some peaks are overlapped and exhibit weaker intensity. Most of the backbone and side-chain resonances were assigned by a standard set of through-bond correlation experiments (See Materials and methods). N-Terminal Met2 and the stretch of residues Lys28, Gln30, His32, Leu33, and Ile34 were not observed in the 1 H- 15 N-HSQC spectrum. Nevertheless, the side-chain resonances of Lys28, Gln30, and Ile34 could be Fig. 1. 1 H- 15 N-HSQC spectrum of pY recor- ded at 600 MHz and 31.5 °C. The protein was in 10 m M sodium phosphate buffer, pH 5.7, and 50 m M NaCl. Assignments are indicated for backbone and side-chain resonances. Question marks correspond to the unassigned Gln and Asn side chains. 5184 K. Ye et al. (Eur. J. Biochem. 269) Ó FEBS 2002 partially assigned in the H(CCO)NH and C(CO)NH spectra, which correlate their resonances to successive residues in the sequence. Resonances of the methyl groups of Leu33 were assigned to two unassigned peaks within the Leu characteristic region in the 1 H- 13 C-HSQC spectrum. The H d1 and H e2 of His32 were assigned in a similar way using the 2D-TOCSY spectrum. All these assignments were self-consistent in the HCCH-TOCSY and 13 C-NOESY spectra. After this, we concluded that three strips of weak peaks observed in the 3D 15 N-NOESY spectrum belong to the spin systems of Gln30, Leu33 and Ile34, and their HN and N signals were assigned despite invisibility in the 1 H- 15 N-HSQC spectrum. Finally, we obtained about 97% of both backbone and side-chain assignments. The C- terminal portion of the protein can degrade over time, resulting in appearance of major and minor degradation products corresponding to residues 1–98 and 1–95, respect- ively. The 1 H- 15 N-HSQC spectrum recorded after degrada- tion of the C-terminal part of pY showed that virtually all peaks corresponding to the residues 2–90 were unchanged, suggesting that the core protein structure is not affected by the C-terminal region. Structure determination The 3D structure of pY was determined using torsion angle simulated annealing protocol with the CNS 1.0 program [25] from a total of 2207 constraints (Table 1), with an average of 24 constraints per residue for amino acids 1–90. Twenty lowest-energy structures were chosen from 30 structures for further analysis. The structural statistics are shown in Table 1. The superposed backbone coordinates of the 20 lowest energy structures are well aligned for residues 1–26 and 36–90 (Fig. 2A). Thus, the core of pY (except residues 27–35) is very well defined by the NMR data. The 23 C- terminal residues are intrinsically disordered as follows from their relaxation properties and their constraints were not included in structure calculations. The overall agreement of the structures with the experimental data is very good, as demonstrated by the small violations (distance violations <0.5A ˚ , no dihedral angle violations > 5°). Excluding the poorly defined region 27–35 and terminal residues, the root mean square deviation (r.m.s.d.) is 0.48 ± 0.14 A ˚ for backbone heavy atoms (N, C a ,C¢), and 1.19 ± 0.15 A ˚ for all heavy atoms. Ramachandran analysis was used to confirm the good quality of the structure. Of the residues, 81.4% have backbone conformation in the most favored regions, and most residues in less favorable conformations are from the poorly defined region (27–35). Use of residual dipolar coupling in the structural calculation The inclusion of residual dipolar couplings (RDCs) has been shown to improve the accuracy, as well as the precision in NMR-based structure determination of proteins [30]. Here we also incorporate RDC information in our attempts to accurately define the orientation of helix a1. As many NMR signals in the loop connecting a1-b2were weak or invisible, only a few NOEs could be observed, this lead to the poor definition of this loop segment. Moreover, the disorder in this region affected preceding helix a1, resulting in a poorly defined orientation for this helix. In the 20 structures calculated without RDC constraints, the C- terminus of a1 flipped away from the b-sheet, such that the interhelical angle between a1anda2 varied over the range of 22.3 ± 2.4°. After incorporating 80 RDC constraints, the interhelical angle became less variable and its value decreased to 16.2 ± 1.4°. As a consequence, the backbone r.m.s.d. of residues 2–26 and 36–88 were also reduced by 0.18 A ˚ . This calculation provides a good example of how long-range orientational constraints can help define aspects of the global geometry under conditions where short-range NOE constraints do not provide sufficient local distance information. The Z-axis of the principal alignment tensor orients along the longest axis of the protein (Fig. 2), consistent with the prediction, which requires the alignment to be mainly Table 1. Structural statistics for 20 structures of pY. CNS energies (kcalÆmol –1 ) a E total 161.19 ± 12.10 E bond 5.33 ± 0.51 E angle 75.42 ± 3.90 E improper 9.56 ± 0.99 E vdw 26.41 ± 4.66 E NOE 27.87 ± 3.93 E cdih 0.05 ± 0.07 E coup 12.34 ± 1.34 E sani 7.39 ± 0.84 r.m.s.d. from idealized geometry Bonds (A ˚ ) 0.002 ± 0.000 Angles (°) 0.388 ± 0.010 Impropers (°) 0.263 ± 0.013 r.m.s.d. from experimental constraints b Distances (A ˚ ) 0.017 ± 0.001 Dihedral angles (°) 0.094 ± 0.053 3 J HNHA coupling (Hz) 0.435 ± 0.023 Residual dipolar coupling (Hz) 0.347 ± 0.019 Ramachandran analysis for 1–90 region (%) c Residues in most favored regions 81.4 Residues in additional allowed regions 16.2 Residues in generously allowed regions 1.3 Residues in disallowed regions 1.1 Pairwise r.m.s.d. for residues 2–26, 36–87 (A ˚ ) Backbone (N, C a ,C¢) atoms 0.48 ± 0.14 All heavy atoms 1.19 ± 0.15 a These values were estimated using CNS 1.0. The final values of the force constants used for the calculations are as follows: 1000 kcal mol –1 ÆA ˚ )2 for bond lengths; 500 kcal mol –1 Ærad )2 for bond angles and improper torsions; 4 kcalÆmol –1 ÆA ˚ )4 for the van der Waals term with the atomic radii set to 0.8 times of their CHARMM values; 50 kcalÆmol –1 ÆA ˚ )2 for NOE-derived and hydrogen-bonding distance restraints; 200 kcalÆmol –1 Ærad )2 for dihedral angle restraints; 1 kcalÆHz )2 for 3 J HNHA restraints; and 0.8 kcalÆmol –1 ÆHz )2 for residual dipolar coupling restraints. b The distance restraints include 408 ambiguous and 1507 unambiguous NOEs, as well as 72 hydrogen-bonding restraints for 36 hydrogen bonds. Unambiguous NOEs comprising 648 intraresidue, 337 sequential (|i–j| ¼ 1), 193 medium-range (1 < |i–j| < 5) and 329 long-range (|i–j| > 4) NOEs. The dihedral angle restraints involve 48 / and 27 v1. There are 65 3 J HNHA restraints, 29 1 D NH and 51 1 D CH residual dipolar coupling restraints. c The values were cal- culated for residues 1–90 using PROCHECK [48]. Ó FEBS 2002 Solution structure of ribosome associated factor Y (Eur. J. Biochem. 269) 5185 induced by steric interaction with the bicelle disc [31]. Because dipolar coupling data cannot define a unique orientation of the interatom vectors, there are four possible orientations of each structure in the ensemble with respect to the principal order axis. Description of structure Figure 2(B) shows the ribbon representation of the calcu- lated pY structure and Fig. 3(A) outlines the pY sequence with secondary structure elements. The protein represents a compact two-layered a/b structure with a babbba topology. The first layer consists of a four-stranded b-sheet, and the second one comprises two a helices. The b1andb2 strands are parallel, while b2, b3andb4 are wound in an antiparallel fashion. The secondary structure elements are defined as follows: b1, Asn3–Ser6; b2, His37–Glu43; b3, Gly46–Thr55; b4, Gly58–Gly64; a1, Thr13–Leu25; a2, Asp69–His88 (Fig. 2). Four turns were identified in the well-defined regions: Glu43–Gly46 and Thr55–Gly58 form type I b-turns; His66–Met69 forms a type VIII b-turn; and Ser6– Met9 adopts a type IV turn. Conformations of all X-Pro peptide bonds were determined to be trans basedonthe observation of the strong H a i À 1 –H d i NOEs. Potential hydrogen bonds were classified based on the identification of slowly exchanging amide protons. All hydrogen bond acceptors, except for Ile15 and Val61, are proposed to involve backbone oxygens from the regular secondary structure. The amide proton of Ile15, located in the beginning of helix a1, can make a potential hydrogen bond to O d of Thr12. Because the amide proton of Val61 points outward towards solvent, it was set to pair with the spatially proximate side-chain oxygen O e1 of Gln83. This proposed hydrogen bond alignment is supported by many NOEs between the side-chain protons of Gln83 and the amide proton of Val61. Backbone mobility In order to investigate the dynamic properties of pY, we collected 15 N relaxation data for the uniformly 15 N-labeled protein. The longitudinal (R1) and transversal (R2) relaxa- tion rates, and 1 H- 15 N heteronuclear NOE values are plotted in Fig. 4 as a function of residue number. No data were obtained for the residues, which did not show amide resonances (Met2, Lys28, Gln30, His32, Leu33, Ile34), show extremely weak intensities (Met9), or involve significant overlapping residues (Ile4, Glu10, Lys42, Lys65, Ala97, Asp102, Val109) in the 1 H- 15 N-HSQC spectrum. The dynamic properties of pY are also outlined in the structure in Fig. 5. Most parts of the protein are fairly rigid. Their 1 H- 15 N NOE values are just below the theoretical maximum of 0.82 (at a 15 N frequency of 60 MHz) (Figs 4 and 5). Some residues in loop b2-b3, around loop b3-b4,andinthe N-terminus exhibit fast motions on the ps-ns timescale, as evident by their lower 1 H- 15 N heteronuclear NOE values (<0.6). The 23 C-terminal residues show significantly lower 1 H- 15 N heteronuclear NOE values and decreased R2 (Fig. 4), indicating that they are completely disordered in the solution. Residues around the long loop a1-b2 and the C-terminal region of helix a1 show higher than average R2 values, making many residues in this region invisible. This suggests that there is significant local conformational exchange on the ms-ls timescale. Such motions may have biological relevance, potentially aiding binding to the ribosome by decreasing the energy cost in the conformation rearrange- ment accompanying the binding process (see role of conserved residues below). Motions spanning a similar range were also reported for the regions responsible for interaction of Bacillus subtilis regulatory protein Spo0F with proteins KinA, Spo0B and RapB [32]. A recent study has directly correlated increased motional flexibility with higher RNA-binding affinity in the double-stranded RNA-binding domain 1 (dsRBD1) of human interferon-induced kinase PKR when compared to dsRBD2 [33]. Slow motions are also found at residues in loop b1-a1, in the turn between b4 and a2, and at residues 52–53 in b3 (Fig. 5). Amino acids 52–53 are spatially close to the loop a1-b2 with distances of 4.6 A ˚ and 6.2 A ˚ from C a atoms of Thr52 and Ile53 to C a atom of Pro36, respectively, and are probably affected by slow motions within this loop. The slowly exchanging hydrogens were generally found within the regular secondary structure elements that do not display fast or slow motion (Fig. 5). The highest protection of hydrogen exchange (P >10 3 ) was observed for residues in helix a2, indicative of this being the most stable structural element. For example, residue Ile77 has the only amide proton still visible after one week of exchange into 2 H 2 O Fig. 2. Structure of pY. (A) Best-fit superpo- sition of the backbone atoms (N, C a ,C¢)ofthe 20 best structures determined for pY residues 1–90. (B) Ribbon representation of a single representative structure. The orientation of the optimized principle order tensor is shown as a frame axes. 5186 K. Ye et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (P >10 4 ). Helix a1, connected to the b-sheet by two flexible loops is less stable than helix a2, as evident from its lower protection factor. Moreover, the exchange protection in this helix shows a bipartite pattern. The amide protons at the buried side of helix a1 are more protected, while the solvent exposed side of helix a1 is less or not protected. This is probably due to the different solvent accessibility and stability of the two faces of helix a1. Sequence conservation ThesequenceoftheE. coli pY protein can be compared with over 50 homologs found in known bacterial genomes, and in Arabidopsis thaliana and Spinacia oleracea plant sequences. A search using FASTA [34] and BLAST [35] revealed only a few hits with significant identity associated with two strains of Salmonella enterica [36,37] (91% identity), Yersinia pestis [38] (79.5% identity), and Pasteu- rella multocida [39] (69.2% identity). Most of the remaining homologs show lower sequence similarity (21–35% identity) with the E. coli pY protein (summarized in Fig. 3A). By contrast, a sequence identity of better than 40% is observed between homologs of ribosomal proteins. The pY-related proteins vary in length and can be grouped into short (78–142 residues) and long (174–229 residues) protein families. The long proteins are similar to pY in their N-terminal segments, and most probably adopt folds similar to the E. coli pY structure. In addition, moderately conserved (25–40% identity) C-terminal exten- sions within the long protein family probably contain additional ribosome binding site(s), as the isolated C- terminal region of the chloroplast-specific pY homolog from Spinacia oleracea can bind E. coli ribosomal particles [8]. Fig. 3. Distribution of the conserved residues in pY. (A) Conserved residues in the sequence of pY. Upper and lower sequences show identical and similar residues, respectively, among 44 pY-related sequences from bacteria. Red, green and yellow residues are 100%, > 80% and > 50% conserved, respectively. Alignment was carried out by FASTA [34] using score matrix BLOSUM 62. Shading was carried out by GENE DOC software (K.B. Nicholas and H.B. Nicholas, Jr., un- published data) using following similarity groups: (1) D, E; (2) N, Q; (3) S, T; (4) K, R; (5)F,Y,W;(6)L,I,V,M.(B)Alignmentof pY with its homolog from plastids of Spinacia oleracea. Residues in red and blue are identical and similar amino acids, respectively. (C) Electrostatic surface of pY. Red and blue colors represent negative and positive poten- tials, respectively. Conserved charged amino acids located on the surface are indicated. (D) Conserved residues (> 80% similar) are represented by cyan balls. Others side chains are shown by sticks colored blue for positive, red for negative, and yellow for polar residues. Ó FEBS 2002 Solution structure of ribosome associated factor Y (Eur. J. Biochem. 269) 5187 Role of conserved residues Most of the conserved residues are located in the N-terminal region of pY (residues 2–26) and towards the end of the well structured segment of the protein (residues 67–90), comprising strand b1, loop b1-a1, helix a1, and helix a2, respectively (Fig. 3A). Many of these conserved amino acids (> 80% similar), as well as a few residues from other regions of the protein, participate in the formation of an extensive hydrophobic core between the inner surfaces of the b-sheet and the two a helices (Fig. 3D). These residues include Ile4 in b1; Met9 and Ile11 in the loop b1-a1; Ile15, Val19 and Leu26 in a1; Leu33 in loop a1-b2; Ile38 and Leu40 in b2; Ile53 in b3; Leu60 and Ala62 in b4; Met69, Ile73, Leu76, Leu80 and Leu84 in a2. Conserved polar amino acids, such as Thr12 and Gln83, may be important for maintaining local conformation, as their side chains are probably involved in potential hydrogen bonds with slow exchanging amide protons: the O d of Thr12 can make a bond with HN of Ile15 in a1, and the O e1 of Gln83 with HN of Val61 in b4. Figure 3(C) shows the molecular surface of pY color coded for electrostatic potential, along with a listing of the conserved solvent accessible residues. Conserved surface features are predominantly localized within the a-helical segments of the protein. The C-terminal parts of the a-helices display a conserved positively charged surface encompassing residues Arg22, Lys25, Lys28, Lys79, Arg82, Lys86 and Lys90. This basic patch can potentially target rRNA upon pY binding to the ribosomal subunit, and the most conserved Lys25, Arg82, Lys86 and Lys90 residues may represent good candidates for involvement in specific RNA recognition. Unlike the C-terminal segments of the a-helices, the N-terminal segment of a2 and the adjacent turn display negatively charged surface patch comprised of Glu75, Glu67 and conserved Asp68 residues. A stretch of six aspartates and glutamates is also present in the unstructured C-terminus of the protein. The unstructured region of pY (C-terminal residues 91–112) does not appear to be important for binding to ribosomes, as E. coli protein YhbH, a homolog of pY, which lacks the 18 C-terminal amino acids, binds equally well to ribosomal subunits [3]. In contrast with the a-helix side of the protein, the solvent exposed face of the b-sheet side of pY consists predomin- antly of polar (Asn3, Thr5, Asn35, His37, Ser41, Thr52, Asn54, Ser63) and a few hydrophobic (Ile34, Ile39, Val48, Val59, Val61) residues (Fig. 3D). Although these residues are poorly conserved, the polar/hydrophobic property of the surface seems to be retained in many Y proteins, for instance, in the distant relative, plastid-specific pY homolog from spinach (Fig. 3B), which can be incorporated into E. coli ribosomes during its in vitro synthesis in bacteria [8]. Remarkably, such a noncharged surface is preserved in the spinach protein despite a doubling of the total number of charged residues. This implies that the solvent exposed face of the b-sheet might be buried within the interior upon binding to ribosomal subunits and contribute to the binding affinity. Comparison with other structures Many proteins with diverse functions exhibit folds related to the two-layered a/b structure of E. coli pY, in which two a-helices pack against a four-stranded b-sheet. However, the E. coli pY structure demonstrates a unique babbba topo- logy, not presented in the SCOP database [40]. Potential structural homologs of pY in the PDB were searched with the program DALI [41]. The search revealed more than 20 Fig. 4. 15 NR1(A),R2(B)and 1 H- 15 N heteronuclear NOE values (C) as a function of the residue number. The secondary structure elements are indicated for reference. Fig. 5. Backbone flexibility in the pY structure. Residues with 1 H- 15 N heteronuclear NOE < 0.6 are red. The residues with fast transverse relaxation rate (R 2 > 16 Hz) are shown in green, and other amino acids are gray. Slow exchanging backbone amides are shown as balls colored according to their protection factor: navy for P > 1000, purple for 1000 > P > 100, sky blue for 10 < P < 100. 5188 K. Ye et al. (Eur. J. Biochem. 269) Ó FEBS 2002 proteins with structural similarity (z score > 2). Half of them are nucleic acid- or nucleotide-binding proteins, with a predominance of RNA-binding proteins amongst them. The best score of 12.2 was found for the recently determined structure of E. coli pY homolog from H. influenzae [10] (see comparison below). Most of the other proteins with structural similarity to the pY proteins can be grouped as follows: (a) proteins with similarity to the double-stranded RNA-binding domain and (b) proteins with similarity to the C-terminal domain of glycyl-tRNA synthetase. The first group includes exclusively RNA-binding pro- teins. These include the dsRBD of Xenopus laevis RNA- binding protein A (Xlrbpa protein) (1 di2, z score 5.4) [42] in complex with 16 bp dsRNA, and dsRBD1 of human interferon-induced protein kinase PKR (1qu6, z score 5.2) [43], which are most similar to pY proteins. Despite the lack of very strong sequence similarity (< 16% identity), the r.m.s.d. values are 3.7 A ˚ (for 64 pairs of C a atoms of Xlrbpa) and 3.2 A ˚ (for 70 pairs of C a atoms of PKR). In addition, the NMR structure of the third dsRBD from Drosophila Staufen protein bound to 18 nucleotide RNA stem-loop [44], which was not picked by DALI program, also exhibits a small r.m.s.d. of 2.4 A ˚ (for 63 pairs of C a atoms). The dsRBD is a 70–80 amino acid long motif, which mediates selective but nonsequence specific double-stranded RNA recognition in a variety of proteins [45]. Proteins can contain a single copy or multiple copies of dsRBDs. Superposition of the pY topology upon the folds of Xlrbpa (Fig. 6), Staufen and PKR revealed good structural simi- larity of pY with these classical dsRBDs, especially in the b sheet and helix a2 segments. Despite the above aspects of overall structural similarity, structural alignment of pY with dsRBDs (Fig. 7) did not reveal high sequence similarity. pY shares with each dsRBDs about 11–16% identical and 6– 11% similar amino acids. When combined, a total of 24% of the pY residues can be found in either of three dsRBDs under comparison and 9% represent similar substitutions. However, only 9% of the amino acids (E43, F47, I53, G64, A72, and L84) match conserved positions of the dsRBD family (Fig. 7), and 11% hit the consensus sequence of dsRBD (not shown) [45]. In turn, most of the conserved residues in pY (68%) do not match amino acids in any of the dsRBD sequences presented here. The pY structure also has a few features that are distinct from dsRBD architecture. First, classical dsRBD has abbba topology, whereas pY has an extra N-terminal b strand. Second, the a helices are virtually parallel in pY, but in dsRBD they make a 25–47° angle. Third, the first a helix in dsRBD is moved along the axis in the C-terminal direction, placing its N-terminal tip in the range of the middle part of the second a helix. Fourth, the part of the b sheet near the C-terminal regions of a helicesisshorterindsRBD, resulting in the a helices extending over the b sheet. Fifth, the b1-b2 loop is longer in dsRBD than the corresponding b2-b3 loop in pY. Consequently, despite overall structural and some sequence similarity between pY and dsRBDs, we are unable to directly address issues related to the common evolutionary origin of the two folds and their potential early divergence. The second group of proteins shares structural similarity with the C-terminal anticodon-binding domain of glycyl- tRNA synthetase. This similarity could have functional implications for pY, given experimental support for pY- mediated translation inhibition of aminoacyl tRNA-ribo- somal A site recognition [2]. However, the C-terminal anticodon-binding domain of glycyl-tRNA synthetase is made of five b strands and three a helices, and therefore, its overall topology is rather different from pY. Moreover, the a helix, which according to the structure of the complex [46] should make most of the contacts to tRNA, has no counterpart in pY. Fig. 6. Structural similarity between pY and dsRBD. The protein structures are shown in the same orientation as on the left panels of Fig. 3(C,D). (A) Superposition of pY (gray) with the dsRBD of Xlrbpa protein (purple) [42] performed with DALI [41]. Protein regions and residues contacting RNA in dsRBD are indicated. Cyan colored amino acids are the same, while orange colored amino acids are different in the complexes of Xlrbpa and Staufen proteins with their RNA targets. (B) Electrostatic surface of the Xlrbpa protein. Red and blue colors represent negative and positive potentials, respectively. Fig. 7. Structural alignment of pY (amino acids 1–89) and dsRBDs. (Ec), protein pY; (Xl), fragment of Xlrbpa [42]; (Pk), the first dsRBD of the human interferon-induced protein kinase PKR [43]; (St), the third dsRBD from Drosophila Staufen [44]. Structure superposition of pY with Xlrbpa and PKR was performed with DALI [41]. Superpo- sition with Staufen was carried out based on homology of the three dsRBDs. Red and blue colors indicate identical and similar residues. Conserved residues (> 50% of identity) of the pY and dsRBD families are shaded. Conservation of pY is as on the Fig. 3(A). Conservation of dsRBDs is adapted from FSSP database [49]. Only residues with C a atomscloserthan4 A ˚ are shown. Note that to present the pY sequence without interruption some unaligned internal amino acids in dsRBDs areomittedinthescheme. Ó FEBS 2002 Solution structure of ribosome associated factor Y (Eur. J. Biochem. 269) 5189 Structure–function relationships Comparison of functional properties of the dsRBD and pY proteins could provide clues towards unraveling their relative structure–function relationships. Two available structures of dsRBD/RNA complexes have revealed details of intermolecular protein-RNA recognition. These involve an X-ray structure of the Xlrbpa fragment complexed with 16 bp dsRNA [42] and an NMR structure of the third dsRBD from Drosophila Staufen bound to a 18 nucleotide RNA stem-loop [44]. There is a minimal change in the conformation of these dsRBDs upon binding their RNA targets. Even disordered loops become only partially ordered in the Staufen complex. dsRBD-dsRNA contacts are mediated by two conserved loops between b1-b2andb3-a2 in these complexes (Fig. 6A). The first loop recognizes predominantly 2¢-OH groups in the minor RNA groove of dsRNA and the second loop interacts with phosphodiester backbone in the major RNA groove. In addition, noncon- served helix 1 makes important contacts with the minor groove in the Xlrbpa complex and with the UUCG tetraloop in the Staufen complex. Noteworthy is half of the amino acids contacting RNA are different in both structures. Despite the apparent resemblance between structures of dsRBDs and pY, their mode of RNA recognition should be different for the reasons outlined below. The longer length of the a1 helix in pY relative to its counterpart in dsRBD proteins makes potential RNA-binding surface less acces- sible in pY for interaction with dsRNA. The shorter length of the b2-b3 loop segment within the pY fold, correspond- ing to the b1-b2 loop in dsRBD, results in the absence of amino acids in pY, which are critical for recognition by dsRBD proteins of their RNA targets (Fig. 6A and 7). In addition, according to the relaxation data, the b4-a2region is very rigid in the pY, although corresponding b3-a2 segment in dsRBDs is flexible in Staufen [44] and PKR [33]. Therefore, it cannot easily undergo necessary motion, required for binding of pY to RNA. Further, there is little similarity in residue distribution within the a helices of the pY and dsRBD proteins. Indeed, the conserved positively charged residues located within the C-terminal regions of the two a helices in the pY protein have no counterparts in dsRBD proteins (Fig. 6B) and conversely, residues K163 and K167 in Xlrbpa, which are rather conserved in dsRBDs from various proteins and appear to be important for RNA recognition [47], are missing in the Y protein. Thus, the pY structure most probably represents a novel functional fold despite elements of structural and sequence similarity and possibly common origin with dsRBD. Related structural research While our work was approaching completion, a similar NMR-based solution structure was published for protein HI0257, a homolog of pY from Haemophilus influenzae [10]. The proteins pY and HI0257 have 64% sequence identity and not surprisingly show very similar structures with the mean backbone r.m.s.d. of 1.5 A ˚ in the core (residue 1–90). A major difference in these two structures lies in the loop a1-b2. In our structure, the loop a1-b2 shows large amplitudes of motions in the ms-ls timescale. Limited structural constraints cause poor definition of this region in the structure. No severe intermediate motion was reported in the HI0257 structure, although some residues, for instance Ile35, show clearly reduced peak intensities in the HSQC spectrum. Therefore, intermediate motion in the loop a1-b2 probably exists in HI0257, but to a lesser extent. Variations in protein sequences probably cause such different dynamic properties between the two highly homologous structures. As expected, all positively charged conserved residues have been found in the structure of HI0257 and, despite larger number of negatively charged residues in the helices of HI0257, both proteins have similar electrostatic surfaces in the presumed RNA-binding site. 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