Tyr51: Key Determinant of the Low Thermostability of the Colwellia

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Tyr51: Key Determinant of the Low Thermostability of Colwellia psychrerythraea Cold Shock Protein Yeongjoon Lee, Chulhee Kwak, Ki-Woong Jeong, Prasannavenkatesh Durai, Kyoung-Seok Ryu, Eun-Hee Kim, Chaejoon Cheong, Hee-Chul Ahn, Hak Jun Kim, and Yangmee Kim Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00144 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Biochemistry

Tyr51: Key Determinant of the Low Thermostability of Colwellia psychrerythraea Cold Shock Protein

Yeongjoon Leea, Chulhee Kwaka, Ki-Woong Jeonga, Prasannavenkatesh Duraia, KyoungSeok Ryub, Eunhee Kimb, Chaejoon Cheongb, Hee-Chul Ahnc, Hak Jun Kimd, and Yangmee Kima*

a

Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea b c

Division of Magnetic Resonance, KBSI, Chungbuk 28119, Republic of Korea

College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, Republic of Korea

d

Department of Chemistry, Pukyong National University, Busan 48547, Republic of Korea

*Corresponding author: Yangmee Kim, Ph.D. Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea Tel.: +822-450-3421; Fax: +822-447-5987; E-mail: [email protected]

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Abbreviations: AFP, antifreeze protein; Bc, Bacillus caldolyticus; Bs, Bacillus subtilis; CPMG, Carr-Purcell-Meiboom-Gill; Csps, Cold-shock proteins; Cp, Colwellia psychrerythraea; Cpse, Corynebacterium pseudotuberculosis; dT6, hexathymidine; dT7, heptathymidine; dT8, octathymidine; Ec, Escherichia coli; hNOE, heteronuclear nuclear Overhauser effect; HSQC, heteronuclear single-quantum coherence; kex, exchange rate; Lm, Listeria monocytogenes; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; OB, oligonucleotide/oligosaccharide-binding; Pbound, population of bound state; pI, isoelectric point; Pi, Psychromonas ingrahamii; RDC, residual dipolar coupling; rmsd, root mean square deviation; RNP, ribonucleoprotein; Ta, Thermus aquaticus; Tm, Thermotoga maritima; Tm, melting temperature

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Biochemistry

ABSTRACT Cold shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of the psychrophile Colwellia psychrerythraea 34H, (Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs—including a five β-stranded barrel—with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10-7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed the formation of a K3–E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 s-1 and 642 s1

), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges

and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

Keywords: cold shock proteins; psychrophile; nuclear magnetic resonance spectroscopy; structure; backbone dynamics; nucleic acid-binding protein

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Introduction Bacteria grow in environments with diverse temperatures, pH, pressures, osmolalities, and salt levels. Temperature is a critical factor for bacterial survival; the highest rates of proliferation are observed at their optimum growth temperatures. Based on environmental adaptations, microorganisms can be grouped into five classes: hyperthermophiles, thermophiles, mesophiles, psychrotrophs, and psychrophiles. Hyperthermophiles, such as Thermotoga maritima, grow optimally at extremely high temperatures, approaching the boiling point of water.1 Thermophiles (e.g., Thermus aquaticus and Bacillus caldolyticus) also grow at temperatures beyond 55 °C.2 Mesophiles, the majority being enterobacteria such as Escherichia coli, preferentially grow at mid-range temperatures of approximately 25–45 °C.35

Listeria monocytogenes, a psychrotroph, also preferentially grows at the aforementioned

moderate ambient temperatures; however, unlike mesophiles, it can survive at temperatures as low as 3 °C, which include typical refrigeration temperatures.6 The optimal growth temperatures of psychrophilic bacteria, such as Colwellia psychrerythraea7 and Psychromonas ingrahamii,8, 9 range from −1 °C to 10 °C in cold marine environments. Since psychrophiles can survive and function at extremely low temperatures (as low as −10 °C), extensive biochemical studies have investigated the mechanisms underlying their cold adaptation, and such enzymes, including psychrophilic lipases, xylanases, and collagenases, are of potential industrial interest.10, 11 Psychrophilic lipases actively hydrolyze triglycerides to free fatty acids and glycerol at low temperatures, and their cold activity is attracting increasing attention because of their potential application in the food industry. Antifreeze proteins (AFPs) are useful natural antifreeze agents in psychrophiles. In fish, AFPs can inhibit ice crystal formation, thereby facilitating their survival in subfreezing seawater. Therefore, AFPs have great biotechnological potential as cryoprotectives and cryopreservatives for biological samples.12, 13

Hence, it is important to understand the fundamental characteristics of the cold-active 4 ACS Paragon Plus Environment

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Biochemistry

proteins of psychrophiles. Enzyme activity decreases below optimum growth temperatures; these conditions deter bacterial metabolic processes, including gene expression, energy production, and biochemical synthesis,14 and this is termed cold shock.15,

16

In addition, protein folding is affected;

consequently, some proteins can get misfolded because of cold denaturation.17 As cell membranes comprise lipids, their fluidity decreases at lower temperatures, unless the composition of the membrane is adapted to the cold environment.18 Finally, ice crystals cause serious damage. To avoid metabolic failure during cold shock, bacteria induce the expression of proteins that support their adaptation to cold temperatures. Cold shock proteins (Csps) are one such type of cold-induced proteins, which act as RNA chaperones by binding to singlestranded nucleic acids and regulate translation. These proteins exist in various bacteria and are highly homologous. Structurally, all Csps contain an oligonucleotide/oligosaccharide-binding (OB) fold, comprising five antiparallel β-strands that assemble to form a β-barrel motif. There are also two nucleic acid-binding motifs, ribonucleoprotein (RNP) 1 and 2, comprising highly condensed regions of basic and aromatic amino acid residues on the surface of Csps. Through these motifs, Csps can bind to single-stranded nucleic acids with relatively high affinity (in the micromolar to nanomolar range). Under low-temperature stress conditions, RNA tends to be stabilized to form nonproductive secondary structures that hinder transcription and translation. Csps act as RNA chaperones, which bind to such RNAs and destabilize the secondary structures to generate the single-stranded form. Thus, they act as transcription anti-terminators and facilitate transcription and translation. Despite the highly conserved structure of Csps, the thermostabilities of these proteins differ markedly, as described by their unfolding or melting temperature (Tm).19, 20 In previous studies, we have investigated the structures and dynamics of a psychrotrophic Csp from Listeria monocytogenes (Lm-Csp) and a thermophilic Csp from Thermus aquaticus 5 ACS Paragon Plus Environment

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(Ta-Csp), using nuclear magnetic resonance (NMR) spectroscopy.19,

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20

However, since the

structure and dynamics of psychrophilic Csps have not been studied yet, the present study aimed to investigate the dynamics of the Csp from the psychrophilic bacterium C. psychrerythraea (Cp-Csp). C. psychrerythraea is an obligate psychrophilic gram-negative bacterium found in ice in the polar oceans; its optimum growth temperature is 8 °C, and it can even grow at −1 °C.7 Since limited information is available regarding cold-shock adaptation of psychrophilic bacteria and the structural features of psychrophilic Csps in comparison with their mesophilic and thermophilic counterparts, we compared the NMR solution structure and dynamics of Cp-Csp to those of thermophilic and mesophilic Csps. Furthermore, we investigated the origin of the increased structural flexibility and lower thermostability of psychrophilic Cp-Csp compared with thermophilic and mesophilic Csps. Since the associations among the structure, dynamics, and thermostability of Csps are expected to correlate with cold shock adaptation in bacteria, understanding these features will provide insights into the fundamental processes underlying the activity of psychrophilic Csps at low temperatures.

Materials and Methods Protein Expression, Isotopic Enrichment, and Purification Wild-type Cp-Csp genes were individually cloned into the pET-11a vector, and the recombinant plasmids transformed into E. coli BL21 (DE3), as described previously.19 To express

15

N and

13

C-labeled Cp-Csps, 1–2 mL of culture was inoculated in 200 mL of M9

minimal medium containing 25 mg/L ampicillin and the isotope-enriched nutrients

15

NH4Cl

and 13C glucose (Cambridge Isotope Laboratories, Andover, MA, USA). Cp-Csp was purified using an anion exchange column (HiTrap QFF, GE Healthcare Bio-Sciences, Uppsala, Sweden) and a reverse phase column (Resource RPC, GE Healthcare Life Sciences, Uppsala, Sweden).19 The yield of protein from 1 L of culture was 1–2 mg. 6 ACS Paragon Plus Environment

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Biochemistry

The mutant Cp-Csp Y51F was generated using the EXchange Site-directed Mutagenesis Kit (Enzynomics, Daejeon, Korea). Polymerase chain reaction (PCR) was performed using 10 fM of the wild-type Cp-Csp gene as a template. A mutagenetic primer pair, 5′caaaccgttgagttcgaagtggggcaag-3′ (forward) and 5′-cttgccccacttcgaactcaacggtttg-3′ (reverse), was used at a final concentration of 0.2 μM of each primer. Deoxyribonucleotide triphosphates (dATP, dGTP, dTTP, and dCTP; 0.2 mM each) and 2 μM of nPfu-Forte DNA polymerase were added. After 30 cycles of denaturation (94 °C, 1 min), annealing (67 °C, 1 min), and elongation (72 °C, 5 min), the mutated gene was successfully amplified.

Measurement of the Binding Affinities of Nucleic Acids to Cp-Csp To measure the binding affinities of nucleic acids to Cp-Csp, we monitored the quenching of the intrinsic fluorescence of the W8 residue of Cp-Csp on nucleic acid binding. The binding constants

of

dT6,

dT7,

and

dT8

were

measured

using a

model

RF-5301PC

spectrofluorophotometer (Shimadzu, Kyoto, Japan) at 25 °C. Cp-Csp protein (10 μM) in 50 mM potassium phosphate buffer (pH 6.0) containing 100 mM KCl and 0.1 mM EDTA was used at a final protein:oligonucleotide ratio of 1:10. The sample was excited at 290 nm, and the light-scattering effects of emission spectra were recorded from 300 to 500 nm. Dissociation constants (Kd) were estimated as described previously.19

Measurement of Circular Dichroism Heat denaturation of Cp-Csp and Cp-Csp-dT7 was observed in circular dichroism (CD) experiments using a J810 spectropolarimeter (Jasco, Tokyo, Japan) and a cuvette with a 1 mm path length. Csps (100 mM) were dissolved in 50 mM potassium phosphate buffer (pH 6.0) containing 100 mM KCl and 0.1 mM EDTA. CD spectra were measured from 190 to 250 nm at 0.1 nm intervals. The mean values derived from data from 10 scans were plotted as the mean 7 ACS Paragon Plus Environment

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residue ellipticity (θ) in deg cm2 dmole-1.21, 22 The melting temperatures of the proteins were determined as the mid-point of the lowest and highest mean residue ellipticities from 5 °C to 80 °C CD signals at 215 nm.

NMR Spectroscopy Experiments and Assignments NMR spectroscopy experiments were performed using the Bruker Avance 500, 700, 800, and 900 MHz spectrometers at the Korea Basic Science Institute (Ochang, Korea), with 2,2dimethyl-2-silapentane-5-sulfonate (DSS) used as an internal chemical shift reference. Csp samples were prepared at a concentration of 0.4–0.5 mM in 50 mM potassium phosphate buffer containing 100 mM KCl and 0.1 mM EDTA (pH 6.0). For backbone assignments, HNCO, HNCACB, and CBCA(CO)NH triple resonance spectra were recorded, and assignments of side chains were obtained from CC(CO)NH, HBHA(CO)NH, H(CCO)NH, and HCCH total correlation spectroscopy (TOCSY) spectra. Backbone assignments were confirmed using 3D 1H-15N and 1H-13C NOESY-HSQC spectra. The HSQC spectrum of 15N-labeled Cp-Csp (0.5 mM) was analyzed using a protein sample at 0.5 mM in 0.3 mL of 9:1 (v/v) H2O/D2O potassium phosphate-buffered solution (50 mM, pH 6.0) containing 100 mM KCl, 0.1 mM EDTA. Chemical shift perturbations in the 1H–15N spectra of Cp-Csp were measured through titration with dT7 at various protein/dT7 ratios (1:0 to 1:1.2). The chemical shift perturbation and changes in the intensities after dT7 binding were measured and used for mapping the binding sites of dT7 in Cp-Csp. All NMR spectra were processed with NMRPipe23 and analyzed with NMRFAM-Sparky24. The first-order temperature coefficients of amide protons in wildtype and mutant Y51F were determined through linear extrapolation from the amide 1H chemical shifts at four different temperatures, 283, 288, 293, and 298 K. Residual dipolar coupling (RDC) between two spins in a backbone amide N-H bond can 8 ACS Paragon Plus Environment

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Biochemistry

be measured by comparing spatially anisotropic dipolar couplings in solution and gel samples. In the gel sample, Cp-Csp molecules were dissolved in a radially compressed polyacrylamide gel, and the strain induced the partial alignment of the protein molecules.25-28 The RDC spectra for solution and gel samples were acquired using doublet-separated HSQC.29 The data were processed with NMRPipe23 and analyzed with NMRFAM-Sparky24.

Determination of Structure Nuclear Overhauser effect (NOE) assignments were carried out using NMRFAMSparky24, and the 3D structure of Cp-Csp was determined using Xplor-NIH based calculations in the PONDEROSA-C/S package30. Thereafter, 20 lowest-energy structures were determined. All violations, including angle and distance, of the best 20 structures were analyzed and refined using PONDEROSA-Analyzer31. The final 20 lowest-energy structures were evaluated using PSVS32. The protein structure figures were generated using PyMOL (http://www.pymol.org).

Backbone Dynamics of Free Cp-Csp and the dT7-Bound Complex We performed NMR spin-relaxation experiments for free Cp-Csp and the dT7-bound complex and analyzed R1, R2, and heteronuclear NOE (hNOE) NMR spectra acquired on a Bruker Avance 500 MHz spectrometer. Longitudinal (R1) spin-relaxation rates were obtained; the relaxation delays were 0.002 (×2), 0.045, 0.1, 0.2, 0.315 (×2), 0.55, 0.8, and 1.00 s. The transverse (R2) relaxation rates were measured with relaxation delays of 0 (×2), 0.0176, 0.0352, 0.0704 (×2), 0.1056, 0.176, 0.2816, and 0.4224 s. The recycle delays for all R1 and R2 relaxation measurements were 2.3 s and 2.0 s, respectively. The heteronuclear cross-relaxation rate was obtained from NOE experiments by interleaving pulse sequences with and without proton saturation. The recycle delay and proton-saturation pulse in hNOE measurements were 4.0 s and 3.0 s, respectively. The offset of the t2 dimension was calibrated to the 1H-NMR 9 ACS Paragon Plus Environment

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chemical shift of water (4.7 ppm), and the offset of the t1 dimension was 121 ppm. Spectral widths were 12 and 36 ppm in the t2 and t1 dimensions, respectively, with 2048 and 256 complex points in the corresponding dimensions. hNOEs were determined from the ratio of peak heights for experiments with and without proton-saturation pulses. R1 and R2 rates were determined by fitting the peak heights in NMRFAM-Sparky.24 Constant-time relaxation-compensated CPMG experiments33 for free Cp-Csp and CpCsp-dT7 were performed using a Bruker Avance 700 MHz spectrometer at the Korea Basic Science Institute. A series of 14 relaxation dispersion data sets were collected at various CPMG field strengths: 0, 50, 75, 100 (×2), 150, 200, 250 (×2), 350, 450, 600, 800, 1000 Hz. The offset of the t2 dimension was calibrated to the 1H-NMR chemical shift of water (4.7 ppm); t1 dimension, 121 ppm. Spectral widths were 12 and 36 ppm in the t2 and t1 dimensions, with 2048 and 256 complex points in the corresponding dimensions, respectively. Twenty-four scans/free induction decay were recorded using a constant time delay of 40 ms and a recycle delay of 2.0 s. Peak heights in NMRFAM-Sparky24 were converted into decay rates, R2eff, for a given CPMG field strength, vcp.34 The two-state model chemical exchange rate (kex) and the population of dT7-bound state (PB) were determined for each residue using NESSY.35 NMR relaxation dispersion data were analyzed with separated CPMG relaxation data sets at different field strengths: 700 and 900 MHz.

Binding model of Cp-Csp and dT7 To propose a docking model of Cp-Csp and dT7, we conducted a molecular docking calculation

using

the

AutoDock

implemented

in

the

YASARA

software

(http://www.yasara.org).36 The NMR structure of Cp-Csp was used as the receptor and dT7 was built as the ligand. All atoms of the receptor were fixed, except the atoms in the side chains of residues showing large changes in the chemical shift perturbation data as well as the spin10 ACS Paragon Plus Environment

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Biochemistry

relaxation data were kept flexible. The simulation box was set around the binding interface near the residues showing large chemical shift perturbations. Every atom in dT7 was flexible during the docking process. After 100 docking runs, the resulting model was subjected to 200 steps of steepest descent minimization in Discovery Studio (Accelrys Software Inc., San Diego, CA, USA.)

Results Sequence Alignment of Bacterial Cold Shock Proteins Fig. 1 shows the alignment of seven bacterial cold shock protein sequences. The top two proteins are psychrophilic cold shock proteins, Cp-Csp from C. psychrerythraea and Pi-Csp from Psychromonas ingrahamii. C. psychrerythraea and P. ingrahamii are known to be the bacteria that can survive at the lowest temperatures, even at −10 °C.7, 8 Furthermore, Lm-Csp is a psychrotrophic cold shock protein expressed by L. monocytogenes. The well-studied BsCsp from Bacillus subtilis and Ec-Csp from E. coli are mesophilic cold shock proteins. Ta-Csp is from the thermophilic bacterium T. aquaticus, which is the source of Taq polymerase. Finally, Tm-Csp is a hyperthermophilic protein from T. maritima. Black regions indicate 100% conserved residues, and boxed regions are 80% conserved residues (Fig. 1). The identity among the sequences of the seven Csps and Cp-Csp is very high and varied, from 50.0% to 75.0%. Distinctively, the thermophilic Csps—Tm-Csp and Ta-Csp—have much higher isoelectric point (pI) values than that of psychrophilic Cp-Csp because they contain more positively charged residues (eleven basic residues), forming stable ionic clusters on the protein surface (Table 1).20, 37 However, Cp-Csp has fewer positively charged residues (six Lys). Notably, it does not have any Arg residue, while other Csps have one to four Arg residues. CpCsp also has fewer negative charges on its surface; it contains only five Glu and four Asp residues. Hence, its pI is slightly higher than that of the psychrotrophic Lm-Csp, which has 11 ACS Paragon Plus Environment

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Figure 1. Alignment of seven prokaryotic Csp sequences. Black, 100% conserved residues; boxed by black lines, 80% conserved residues. The red boxes represent the two ribonucleoprotein (RNP) sites, RNP 1 and RNP 2.

five more negative charges (seven Glu and five Asp) but a similar amount of positive charges (two Arg and four Lys). The nucleic acid-binding motifs RNP 1 and 2, enclosed in red boxes, are highly conserved in all bacterial Csps and contain numerous hydrophobic residues and positively charged residues, which are essential for their interactions with nucleic acids. The aromatic residues are generally conserved among all Csps (Fig. 1). The number of aromatic residues (Trp-Phe-Tyr) are 1-4-2 for both psychrophilic Cp-Csp and Pi-Csp, 1-7-0 for Lm-Csp, 1-7-0 for Bs-Csp, 1-61 for Ec-Csp, 1-6-1 for Ta-Csp, and 2-5-1 for Tm-Csp. In Cp-Csp, four of seven aromatic residues, W8, F15, F17, and F28, are exposed on the nucleic acid-binding surface, and three, W8, F17, and F28, are conserved in all seven Csp sequences analyzed. The type of aromatic residues also varied in the RNP sites. Interestingly, in the thermophilic proteins (Tm-Csp and Ta-Csp), the third amino acid in the RNP 1 motif is Tyr, rather than a Phe residue. In general, the last residue of RNP 2 is Phe; however, it is Trp in the hyperthermophilic Tm-Csp and His in the psychrophilic Csps, Cp-Csp and Pi-Csp. These subtle differences in the hydrophobic stacking interactions in the nucleic acid-binding region reduce the binding affinity of psychrophilic Csps. In contrast, the aromatic side chains of F9 and Y51 among seven aromatic residues participate in hydrophobic core packing in psychrophilic Csps. In this β 4 site, only psychrophilic Csps contain Tyr51 instead of the conserved Phe51 in mesophilic and 12 ACS Paragon Plus Environment

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Biochemistry

Table 1. Melting and growth temperatures of bacterial cold shock proteins Tm(˚C) of Free Csp Growth Temperature (dT7 complex) (Optimum) (˚C)

Classification

Bacterium

Csp

pI

Hyperthermophile

T. maritima

Tm-Csp

6.73

8538

55 ~ 90 (80)1

Thermophile

T. aquaticus

Ta-Csp

6.73

7620

50 ~ 80 (70)2

E. coli

Ec-Csp

5.58

6339

10 ~ 49 (37)3

B. subtilis

Bs-Csp

4.54

5038

11 ~ 524 (43-465)

Psychrotroph

L. monocytogenes

Lm-Csp

4.45

40 (52)19

3 ~ 45 (30-37)6

Psychrophile

C. psychrerythraea

Cp-Csp

5.11

37 (50)

-1 ~ 10 (8)7

Mesophile

thermophilic Csps. Y40 is located in the long flexible surface loop 3 region between β3 and β4 strands, which is distal to the nucleic acid-binding region.

Oligonucleotide-Binding Affinities of Nucleic Acids to Cp-Csp The binding affinity of nucleic acids to Cp-Csp was measured by monitoring the quenching of the fluorescent indole side chain of the W8 residue at the nucleic acid-binding interface.19 According to previous reports about the binding preferences for various nucleotides of Csps from B. subtilis, B. cardolyticus, and L. monocytogenes, Csps have higher affinity to oligonucleotides including thymidine compared to those including cytosine or uracil.19, 40, 41 Oligonucleotides consisting of seven thymidines (dT7) showed the highest binding affinity among dT6, dT7, and dT8 for all Csps. The binding affinities of single-stranded DNA to BsCspB or Lm-Csp are markedly higher than those of single-stranded RNA to these proteins, probably because the additional methyl group of thymine, relative to uracil, mediates the tight binding of DNA to Csps through hydrophobic interactions. The dissociation constants of CpCsp for hexathymidine (dT6), heptathymidine (dT7), and octathymidine (dT8) at 25 °C were 1.6 × 10-6, 3.8 × 10-7, and 1.1 × 10-6 M, respectively. Since the thermostability of Cp-Csp was very 13 ACS Paragon Plus Environment

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low, further experiments were performed using dT7 with the highest binding affinity to stabilize the structure of Cp-Csp. Cp-Csp displayed a lower binding affinity to dT7 than that of the psychrotrophic Lm-Csp (1.3 × 10-7 M),19 while Ta-Csp displayed a stronger binding affinity for dT7 at 1.2 × 10-8 M.20 Similarly, the thermophilic Bc-Csp has a strong binding affinity for dT7 (0.9 × 10-9 M).41 Since thermophilic Csps have more positively charged residues, they tend to have stronger binding affinities for nucleic acids than psychrophilic Csps do because of electrostatic interactions with the negatively charged backbones of the nucleic acids.

Melting Temperatures of Bacterial Csps The thermostabilities of bacterial Csps are correlated with the lower limits of growth temperature for those microorganisms. Although bacterial Csps share a large degree of sequence homology and structural similarity, they exhibit drastic differences in thermostability. We determined the melting temperature of Cp-Csp using CD spectroscopy. The CD spectra of Cp-Csp exhibited certain distinctive features, with positive intensities from 215 nm to 230 nm, because of numerous aromatic rings of the single Trp, four Phe, and two Tyr residues in CpCsp (Fig. 2a). These were strong positive contributions, resulting from π-π* transitions of aromatic side chains overlapping with a broad negative peak at approximately 215 nm from the n-π* transition of the anti-parallel β-sheets, leading to minimum and maximum intensities at approximately 210 and 222 nm, respectively.42-46 These features are common among the majority of Csp CD spectra, since all Csps have more than seven aromatic residues among their total of 66–73 residues,

which represents

an exceptionally

high ratio. 43

The

melting temperature (Tm) of the transitions between denatured and native states were monitored via the changes in the CD signal at 215 nm, and the fraction of native protein determined, assuming that Cp-Csp has a two-state model, with denatured and native states. Analysis of the 14 ACS Paragon Plus Environment

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Figure 2. CD spectra of bound and free Cp-Csp. (a) CD spectra of intact free Cp-Csp (●) and Cp-Csp-dT7 (○) in 50 mM potassium phosphate containing 100 mM KCl and 0.1 mM EDTA (pH 6.0). (b) Temperature induced a change in the folding of free Cp-Csp (●) and dT7-bound Cp-Csp (○), monitored as changes in the mean residue ellipticity at 215 nm. thermal denaturation curves obtained from 5 °C to 80 °C indicated that the Tm of Cp-Csp is 37 °C and that of Cp-Csp-dT7 is 50 °C, implying that the binding of dT7 stabilizes the structure of Cp-Csp. Table 1 shows the tendency of increasing melting temperature and optimum growth temperature from psychrophiles to hyperthermophiles. Mesophilic Bs-Csp and Ec-CspA have melting temperatures of 50 °C and 63 °C, respectively.39 Thermophilic Bc-Csp and Ta-Csp can tolerate temperatures beyond 70 °C.20, 38 Tm-Csp, from a bacterium that grows optimally at 80 °C, is even more thermostable, with a Tm of 85 °C.38 In contrast, the Tm of the psychrotrophic protein Lm-Csp is 40 °C, which is lower than those of mesophilic Csps, as determined in our previous study.19 Even with high sequence homology, psychrophilic Cp-Csp has markedly lower thermostability and optimum growth temperature (8 °C) than its mesophilic and thermophilic counterparts. Therefore, the present study aimed to elucidate the reasons underlying the differences in thermostability in various Csps and to investigate the important structural and 15 ACS Paragon Plus Environment

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dynamic characteristics of Cp-Csp.

Tertiary Structure of Cp-Csp We next determined the three-dimensional structure of Cp-Csp using NMR spectroscopy. Structural statistics for the 20 lowest-energy structures of Cp-Csp are summarized in Table 2. Four different types of input restraints were used for the calculation (Table 2). All prokaryotic Csps have conserved structural features, including five antiparallel β-strands forming a β-barrel structure containing an OB fold. Nevertheless, these highly conserved proteins have significant differences in their detailed features, including in close-range ionic interactions, hydrophobic packing, and the length of the β-strands. The structure determined through NMR spectroscopy revealed that Cp-Csp has a closed β-barrel consisting of five β-strands: β1, K3–N10; β2, F15– T19; β3, L27–H30; β4, T48–G54; β5, C61–A67 (Fig. 3a). The antiparallel β-pleated sheets may be stabilized by hydrophobic interactions and backbone hydrogen bonds within the β-sheets. The hydrophobic core contains 10 hydrophobic residues: V6, F9, I18, L27, V29, L43, V49, Y51, A62, and V65. Psychrophilic Cp-Csp has only one salt bridge, K7–D26, on its surface. This ionic interaction is well conserved among the bacterial Csps. Other Csps from organisms with the higher optimum growth temperatures have at least one more salt bridge in addition to this conserved interaction. Thermophilic Csps tend to have extensive salt bridges on their surfaces. The lack of electrostatic interaction indicates that the Cp-Csp structure is more flexible than those of other Csp homologs. The superposition of 20 structures is presented in Fig. 3b. As listed in Table 2, the average root-mean-square deviation (rmsd) of all backbone atoms was 0.7 Å, and that of ordered residues (K3–H30 and T48–A67) was 0.4 Å. The highly flexible loop 3, H31–Q47, exhibited notably large deviations, and the rmsd of this region was 1.1 Å, almost threefold that of ordered

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Biochemistry

Figure 3. The structure of Cp-Csp determined through NMR spectroscopy. (a) The 3D structure of Cp-Csp (PDB accession: 5XV9; BMRB ID: 36102). The ionic interaction is represented as a yellow-dotted line. (b) Superposition of the backbones of 20 model structures. (c) The hydrophobic core packing of Cp-Csp. Two hydrophobic residues, I34 and V53, denoted in red, are excluded from the packing in Cp-Csp, whereas these are core residues in mesophilic and thermophilic Csps. The aromatic side chain of Y51, whose phenol ring is oriented toward the solvent, is denoted in orange. The structure is rotated along the y-axis by 90° (left) and 180° (right) along the z-axis, compared with that shown in (a) to denote the residues constituting the hydrophobic cavity.

regions (Table 2). Ramachandran plots for the 20 structures revealed that over 80% of residues were positioned in the most favored regions, and no residues were outside the permitted regions. Furthermore, there were no average violations for any of the determined structures in either distance or angle constraints. The hydrophobic cavity is shown in Fig. 3c. The hydrophobic packing of the 10 residues constituting the hydrophobic cavity was much looser than that of thermophilic Csps (12 residues)20, 37 and mesophilic Csps (12 residues)47. In Cp-Csp, I34 and V53 are exceptionally excluded from the hydrophobic cavity; therefore, psychrophilic Cp-Csp has looser hydrophobic packing than that of its mesophilic and thermophilic counterparts, resulting in its lower thermostability and more flexible structure. NOE peaks provided in Fig. S1a are possibly responsible to the outward orientations of I34 and V53 side chains as shown in Fig.S1b. The side chain of the conserved I34, which is located in the hydrophobic cavity in other thermophilic and mesophilic Csps, appears to be excluded from the packing in Cp-Csp because of the high degree of flexibility around this residue. The negatively charged E33 residue, 17 ACS Paragon Plus Environment

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Table 2. Structural statistics of the 20 lowest-energy structures of Cp-Csp Restraints a Total Conformationally restricting distance constraints Short Range [(i– j) 5] Dihedral angle constraints Phi Psi Residual dipolar coupling constraints Hydrogen-bond constraints Xplor-NIH pseudo-potential energy [kJ/mol] b Average rmsd to the mean Xplor-NIH coordinates [Å] b Backbone atoms (all residues / ordered residues c) Heavy atoms (all residues / ordered residues c) Ramachandran plot summary from PROCHECK [%] b Most favored regions Additionally allowed regions Generously allowed regions Disallowed regions Average number of violations per Xplor-NIH conformer d Distance constraint violations (> 0.2 Å) Angle constraint violations (> 10°)

842 228 88 349 51 53 49 24 2417 0.7/0.4 1.2/0.9 92.4 7.6 0.0 0.0 0 0

a

The 3D structure of Cp-Csp was calculated using Xplor-NIH based calculation in PONDEROSA-C/S.30 The final 20 lowest-energy structures were evaluated using PSVS.32 c Ordered residues: K3-H30, T48-A67 d All violations of the 20 best structures were analyzed using PONDEROSA-Analyzer.31 b

immediately before the I34, is conserved only in psychrophilic Cp-Csp, while all other Csps exclusively contain alanine residues at this position (Fig. 1). As E33 may interact with the solvent more readily than the conserved alanine in other Csps, it can contribute to high flexibility at the beginning of loop 3 in this protein. Compared to those of mesophilic and thermophilic Csps, loop 2 and loop 3 of Cp-Csp were longer and more flexible. Therefore, both the number of hydrophobic core residues and the lengths of the flexible loop region between β-strands also contribute to the thermal stability of Csps. An additional unique feature of the hydrophobic packing of Cp-Csp is that psychrophilic 18 ACS Paragon Plus Environment

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Csps, including Cp-Csp, contain Y51 in the middle of the β4 strand, while mesophilic and thermophilic Csps contain F51. The aromatic moiety of Y51 in Cp-Csp appears to be displaced from the middle of the core, which disrupts the tight packing of the hydrophobic side chains within the core.

Chemical Shift Perturbations of Cp-Csp upon dT7 Binding By comparing the backbone amide 1H and

15

N chemical shifts of free Cp-Csp and Cp-

Csp-dT7, we determined its nucleic acid-binding site. Fig. 4a shows an overlay of the 15N–1H HSQC spectra of free Cp-Csp and Cp-Csp-dT7; the light blue arrows indicate peaks of significant chemical shift variation on dT7 binding. Chemical shift perturbation of backbone amide protons mainly highlighted the residues proximal to the RNP 1 and RNP 2 domains. The aromatic residues in Cp-Csp, including W8, F15, F17, F28, and H30, develop hydrophobic interactions with the bases of dT7 (Fig. 4b). Furthermore, the residues showing large chemical shift perturbations were proximal loop 3 and loop 4 regions. A41 located in the loop 3 region exhibited large chemical shift perturbations, implying the occurrence of conformational rearrangements distal to the binding interface. K58 located in the loop 4 region may also have electrostatic interactions with nucleic acid phosphate groups. Fig. 4c shows the peak traces of the K13 and F28 residues of Cp-Csp titrated with dT7 at a 1:1 ratio in 1H–15N HSQC. When dT7 was added, the peaks showed gradual unidirectional shifts until saturation, implying that the exchange rate between the free and bound states is greater than the NMR time scale. The Hε1-Nε1 in the indole side chain of the W8 nucleic acid-binding site exhibited a large chemical shift perturbation (0.772 ppm), as shown in Fig. 4b, implying that the indole side chain may participate in the interaction with dT7. These results suggest that the side chains of W8, N10, residues at RNP 1 and RNP 2 domains, and K58 in Cp-Csp may be the key residues for interaction with dT 7 through 19 ACS Paragon Plus Environment

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Figure 4. Chemical shift perturbations of Cp-Csp on dT7 binding. (a) Overlay of the 1H−15N HSQC spectra of free Cp-Csp (black) and Cp-Csp-dT7 (red) at a 1:1 Csp:dT7 molar ratio. Blue arrows indicate residues exhibiting chemical shift variations of >0.2 ppm on dT7 addition. (b) Chemical shift perturbation upon addition of dT7. The weighted average of the differences in 15 N and 1H chemical shifts for each residue was calculated as follows17: ∆δ = √∆δ𝐻 2 + 0.2∆δ𝑁 2 The perturbation for the Hε1-Nε1 of Trp8 (blue bar) is included. (c) Peak traces in the HSQC spectra for the dT7 titration, showing the signal changes for K13 (left) and F28 (right) after titration with dT7 at ratios of 0:1 (black), 0.1:1 (yellow), 0.3:1 (sky blue), 0.5:1 (blue), 0.7:1 (green), 1:1 (red), and 1.2:1 (magenta). hydrophobic interactions or electrostatic interactions. Previous studies have shown that Csps have almost invariant structures upon binding to dT7.19, 20, 40 We also observed very similar NOE patterns for Cp-Csp-dT7 to those of free Cp-Csp. Therefore, we did not determine the structure of the Cp-Csp-dT7 complex in this study, similar to the cases of Lm-Csp and Ta-Csp.19, 20

Instead, we investigated the dynamic properties of free Cp-Csp and the Cp-Csp-dT7 complex,

which may provide important information about the interactions between Cp-Csp and dT7.

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Chemical Shift Perturbations upon Y51F Mutation To investigate the role of Y51, we induced a Y51F substitution in Cp-Csp via site-directed mutagenesis. E21, D22, and G23, which are solvent-exposed residues at the beginning of loop 2, caused significant chemical shift perturbations in the 1H-15N HSQC spectra of the Y51F mutant (Fig. 5a). The phenolic hydroxyl group of Y51 is close to backbone amide protons of E21 (3.7 Å). Therefore, the hydroxyl group of Y51 may form a weak hydrogen bond with the backbone amide protons of E21, as shown in Fig. 5b. In other Csps, the aromatic side chain of Phe at this position mediates compact packing in the hydrophobic cavity. Because of the side chain of Y51 being withdrawn from the core, the side chain of V53 may protrude toward the surface along with it. Therefore, the side chain of V53, which is also located in the hydrophobic cavities of other Csps, does not participate in hydrophobic packing, and this loosely packed hydrophobic core significantly reduces the thermostability of Cp-Csp. We also determined the melting temperature of the Y51F mutant. Although wild-type and Y51F displayed similar folding patterns in CD spectra (Fig. 5c), its Tm increased from 37 °C to 43 °C, confirming that Y51 disrupts the compact hydrophobic packing of psychrophilic Csps. Furthermore, to investigate the structural stability by monitoring the temperature dependence of the chemical shift of each residue, we determined and compared the first-order temperature coefficients of wild-type and mutant Y51F through linear extrapolation from the amide 1H chemical shifts at four different temperatures, 283, 288, 293, and 298 K. The result validated that mutant Y51F is less sensitive to temperature change than the wild-type protein. The overall average of temperature coefficients increased from −3.01 to −2.00 ppb/K owing to the Y51F substitution. Since Y51F enhances hydrophobic packing, the average of temperature coefficients of 10 packing residues also increased from −2.07 to −1.09 ppb/K. The number of amide protons with temperature coefficients lower than −5 ppb/K decreased from 21 ACS Paragon Plus Environment

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Figure 5. Chemical shift perturbations in the 1H–15N HSQC spectra of Cp-Csp upon introducing the Y51F substitution via site-directed mutagenesis. (a) Overlay of the 1H−15N HSQC spectra of wild-type Cp-Csp (black) and mutant Y51F Cp-Csp (red). Blue arrows indicate residues showing significant chemical shift variations, and the bar graph shows the chemical shift perturbation in parts per million after mutagenesis. (b) View of the region showing the interactions between the phenolic hydroxyl group of Y51 (orange) and backbone amide protons of E21, D22, G23, and K3 (yellow). (c) CD spectra of wild-type (●) and mutant Y51F Cp-Csp (○) in undenatured states. Temperature induced a change in the folding of wildtype Cp-Csp (●) and Y51F Cp-Csp (○), detected as alterations in the mean residue ellipticity at 215 nm. (d) First-order temperature coefficients of amide protons in wild-type and mutant Y51F. E21 is highlighted in a red circle, and the red line indicates −5 ppb/K. The temperature dependence on amide proton chemical shift changes of E21 for both wild-type and mutant Y51F is shown.

17 to 10, implying that the Y51F mutant has more stable folding and less solvent-exposed 22 ACS Paragon Plus Environment

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Biochemistry

amide protons than those of the wild-type protein.48 Surprisingly, the temperature coefficient of E21 increased dramatically from −5.44 to −2.21 ppb/K (Fig. 5d), implying that E21 becomes more stabilized by the Y51F mutation. Furthermore, large chemical shift perturbations of K3 and G4 were also observed. These results imply that the removal of constrained hydrogen bonding between Y51 and E21 owing to the Y51F mutation may facilitate the formation of a K3–E21 salt bridge. In addition to more compact hydrophobic packing, this might be the key reason for the increased melting temperature from 37 °C to 43 °C through the Y51F mutation in Cp-Csp.

Backbone Dynamics of Cp-Csp and Cp-Csp-dT7 To investigate the dynamic properties of Cp-Csp at lower temperatures, closer to the optimum growth temperature of C. psychrerythraea, we performed backbone relaxation experiments on Cp-Csp at three different temperatures—25 °C, 15 °C, and 5 °C—using a 500 MHz NMR spectrometer (Fig. 6). The average R1, R2, and hNOE values of free Cp-Csp were 2.34 ± 0.25 s-1, 5.68 ± 2.25 s-1, and 0.49 ± 0.14 units at 25 °C; 2.31 ± 0.19 s-1, 6.28 ± 1.53 s-1, and 0.60 ± 0.13 units at 15 °C; and 2.05 ± 0.12 s-1, 7.93 ± 1.43 s-1, and 0.64 ± 0.14 units at 5 °C, respectively. Spin-relaxation data for Cp-Csp revealed large R2 values for the backbone N-H of the K7 (14.79 s-1) and W8 (16.86 s-1), residues, and these rates gradually decreased with decreasing temperatures. As expected, higher hNOE values at lower temperatures indicated increased structural rigidity. In particular, the average hNOE value for the loop 3 region from H31 to Q47, which has a very flexible structure, increased slightly from 0.41 ± 0.17 at 25 °C to 0.59 ± 0.16 at 5 °C, implying that this long loop region becomes more rigid at lower temperatures, while still remaining very flexible. Markedly, in the loop 4 region (Q55 to P60), high loop flexibility was retained, even at 5 °C, which is close to the optimum growth temperature of this bacteria. 23 ACS Paragon Plus Environment

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Figure 6. Results of backbone relaxation experiments to determine the R1, R2, and hNOE values for Cp-Csp. R1, R2, and hNOE values were determined at various temperatures: (a) 25 °C, (b) 15 °C, and (c) 5 °C. The missing points in the plots are Pro20 and Pro60.

The dynamic properties of free Cp-Csp were compared with those of complexes with nucleic acid (Cp-Csp-dT7) (Fig. 7a). The average R1, R2, and hNOE values of free Cp-Csp were 2.34 ± 0.25 s -1, 5.68 ± 2.25 s -1, and 0.49 ± 0.14 units, respectively, whereas the corresponding values for dT7-bound Cp-Csp were 2.42 ± 0.23 s-1, 5.72 ± 0.98 s-1, and 0.65 ± 0.14 units, respectively. The average R2/R1 ratio for free Cp-Csp was 2.41 ± 0.83, whereas that for bound Cp-Csp was 2.36 ± 0.37, implying that both free and bound Cp-Csp exist as monomers. Cp-Csp spin-relaxation data showed that large R2 values for the backbone amides of K7 and W8 in free form may result from the conformational exchange (R ex) of the protein at microsecond-to-millisecond time scales. Upon dT7 binding in complex form, R2 values of K7 (14.79 to 7.15 s−1) and W8 (16.86 to 8.02 s−1) decreased, since they are located at nucleic acid-binding sites. This drastic reduction in R2 values upon binding suggests that electrostatic 24 ACS Paragon Plus Environment

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Biochemistry

Figure 7. Results of backbone relaxation experiments to determine the R1, R2, and hNOE values for the free and nucleic acid-bound forms of various Csps. R1, R2, and hNOE values for the free form (●) and dT7-complex (○) of (a) psychrophilic Cp-Csp (Pro20 and Pro60 are missing), (b) psychrotrophic Lm-Csp19 (Pro58 is missing), and (c) thermophilic Ta-Csp20 (Pro24, Pro53, and Pro60 are missing) at 25 °C. or hydrophobic interactions with a nucleic acid may stabilize the structure of Cp-Csp upon dT7 binding. We also compared the spin-relaxation data of Cp-Csp with those of Lm-Csp and Ta-Csp, which were investigated previously,19, 20 revealing that the overall patterns were similar (Fig. 7). Similar to Cp-Csp, Ta-Csp had high R2 values proximal to the β1 and β2 strands and in the loop in between them, while more conformational exchanges occurred in Lm-Csp throughout the protein. Comparing the average R2 values of three different Csps, all Csps displayed drastic reductions in R2 rates upon dT7 binding. Unlike other Csps, psychrophilic Cp-Csp showed increased R2 rates in the turn regions of loop 1 (G14) and loop 3 (A41) on binding to dT7. (Fig. 7). It appears that these loop regions, distal to the nucleic acid-binding sites, are structurally 25 ACS Paragon Plus Environment

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rearranged on binding to dT7. The hNOE values of residues in the β3 and β4 strands of Cp-Csp increased more markedly than those of Lm-Csp (Fig. 7b)19 or Ta-Csp (Fig. 7c)20 on dT7 binding. In Cp-Csp, the average hNOE values for β3 and β4 strands in the unbound state were 0.57 and 0.56, respectively, and they increased to 0.74 and 0.73, respectively, on dT7 binding. For Lm-Csp, the average hNOE value of β3 increased from 0.66 to 0.72, while that of β4 increased from 0.64 to 0.69,19 and for Ta-Csp, the corresponding increases were 0.62 to 0.72 for β3 and 0.61 to 0.70 for β4.20 This implies that the structural rigidity of the β3 and β4 strands in Cp-Csp increases their sensitivity to dT7 binding than their homologous counterparts. However, dT7 binding had a rather minor effect on the hNOE values of the highly flexible loop 3 region of Cp-Csp compared with the β3 and β4 strands. Residues in the flexible loop 3 and loop 4 regions of Cp-Csp exhibited markedly lower hNOE values than those of thermophilic Ta-Csp and psychrotrophic Lm-Csp. The hNOE value of the loop 3 of free CpCsp increased but remained extremely low after dT7 binding (0.27 to 0.57) in comparison with those of Lm-Csp (0.52 to 0.67) and Ta-Csp (0.51 to 0.63).19, 20 In loop 3, Cp-Csp has two continuous G37 and G38 residues with extremely low hNOE, 0.20 and 0.09, respectively. Furthermore, in contrast to other Csps, Cp-Csp has A41 (hNOE value in the dT7-bound state is 0.37) instead of conserved basic residues such as K39 in Lm-Csp and R39 in Ta-Csp. These basic residues share electrostatic interactions with nearby negatively charged residues, resulting in higher hNOE values in the middle of long loop 3 (K39 in Lm-Csp-dT7, 0.52; R39 in Ta-Csp-dT7, 0.72). Similarly, the rigidity of loop 4 increased from 0.28 to 0.48, since K58 binds nucleic acids via electrostatic interactions (0.15 to 0.46). These data are concurrent with the observed large chemical shift perturbations of G56 and K58 upon dT7 binding. Therefore, the flexibilities of the loop 3 and loop 4 regions are higher in psychrophilic Cp-Csp than in other Csps, and these 26 ACS Paragon Plus Environment

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Biochemistry

flexibilities contribute to relative reductions in protein thermostability.

Microsecond-to-Millisecond Time Scale Motions in Cp-Csp and Cp-Csp-dT7 Motions in Cp-Csp on the microsecond-to-millisecond time scale were confirmed through constant-time relaxation-compensated Carr-Purcell-Meiboom-Gill (CPMG) experiments (Fig. 8a).33, 34, 49 Differences in R2eff values (ΔR2eff) were calculated from the long (νcp = 100 Hz) and short (νcp = 1000 Hz) interpulse delays, where cp is the CPMG field strength. Consistent with the backbone spin-relaxation result, the backbone N-H of K7 and W8, which exhibited high R2 values, were identified to undergo chemical exchange on a microsecond-to-millisecond time scale, with large ΔR2eff values (>10 s-1), and these rates decreased on dT7 binding (Fig. 8b). Interestingly, we identified additional residues that exhibited large conformational exchanges in the microsecond-to-millisecond time scale, although they did not show high transverse relaxation rate R2 values. ΔR2eff values of >10 s-1 were observed for F9, S11, F15, G16, F28, V29, H30, Q35, A41, L43, A44, V53, K58, and V66. Some of these residues interact with single-stranded nucleic acids, while others are located in the loop regions, or have solventexposed hydrophobic side chains. F15 (14.00 s-1), F28 (13.49 s-1), H30 (13.49 s-1), and K58 (10.02 s-1) interact with nucleic acids, and these motions were not observed on dT7 binding (Fig. 8b). F9 (12.48 s-1) and V29 (10.03 s-1) are involved in hydrophobic packing, rather than the binding interactions. S11 (14.00 s-1) is the first residue of loop 1, located immediately after the β1 strand. Q35 (15.93 s-1), A41 (13.24 s-1), L43 (17.71 s-1), and A44 (14.54 s-1) are located in the highly flexible loop 3 region. V53, which is exposed to the solvent only in the psychrophilic Cp-Csp, has a ΔR2eff value of >10 s-1. The hydrophobic side chain of V66 (12.58 s-1) is also exposed to the surface. After dT7 binding, most of these residues lost backbone motion, as they were stabilized by the binding interactions (Fig. 8c). dT7 binding reduces the slow time scale motion at the 27 ACS Paragon Plus Environment

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Figure 8. Constant-time relaxation-compensated (CPMG) experiments to investigate microsecond-to-millisecond time scale motions of Cp-Csp. Chemical exchanges in (a) unbound-form Cp-Csp and (b) dT7-bound form Cp-Csp at 700 MHz and 25 °C. The exchanges were measured as differences in relaxation rates at two separated interpulse delays, 100 Hz and 1000 Hz. (c) Overlay of CPMG decay for three residues at the nucleic acid-binding interface. Data were acquired through 700 MHz NMR for unbound Cp-Csp (●) and Cp-Csp-dT7 (○). Relaxation dispersion data obtained through 900 MHz NMR for unbound Cp-Csp (▼) are shown in red. Solid lines indicate fitted curves.

nucleic acid-binding site. In the unbound state, the aromatic rings of F15 at RNP 1 and F28 at RNP 2 are exposed to the solvent, and the two conformations may be exchanged, while in the dT7-bound state, nucleic acid binding stabilized the bound conformations. In contrast, some of the residues in loop 3, such as A41, distal to the nucleic acid-binding site, still exhibited slow exchanges on dT7 binding. A41 also showed a large chemical shift perturbation on dT7 binding. Only psychrophilic Csps have an A41 residue, rather than a basic residue, which exhibited a chemical exchange in the presence of dT7 in the CPMG relaxation experiment (Fig. 8b). In the tertiary structure of the free form of Cp-Csp, the hydrophobic side chain of A41 orients toward the interior of the turn region of loop 3. In other Csps, this site is a basic residue and is exposed to solvent. It appears that the binding interactions of Cp-Csp with dT7 potentially cause 28 ACS Paragon Plus Environment

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structural rearrangement and exposure of the side chain of A41 toward the solvent, inducing microsecond-to-millisecond timescale motions at this residue. The relaxation dispersion data at 700 and 900 MHz NMR for W8, F15, and F28, which are located at the dT7-binding site, are shown in Fig. 8c. The exchange rates (kex) between free and nucleic acid-bound states, and the population in each state were determined through NMR dispersion data analysis, using two separate data sets of free form at two different field strengths, 700 MHz and 900 MHz.27, 50-52 They were calculated using NESSY.35 We assumed that the major conformation was the free state; and the minor conformation, the bound state. The average exchange rates (kex) were 754 s-1 and 642 s-1 for RNP 1 and 2 regions, respectively. The average population of the bound state (Pbound) was 3% for both nucleic acid-binding motifs. Interestingly, the C-terminal region of the β1 strand (K7 and W8) showed high exchange rates, on an average, 6347 s-1, with 4% population in the bound state. These motions in the binding interface are probably associated with the accommodation of nucleic acids on the protein surface.

Binding model of Cp-Csp and dT7 NMR experiments revealed that the residues near the dT7 binding exhibited remarkable changes after dT7 binding, such as a large chemical shift perturbation (>0.2 ppm), large decrease in R2 rates (>5 s-1), or large decrease in ΔR2eff (>9 Hz). hNOE values of all these residues are increased by more than 0.1. The backbone amide N-H peaks in the 1H-15N HSQC spectrum of N10, G14, F15, G16, F17, F28, V29, H31, S32, A41, G56, and K58 showed significantly large chemical shift perturbations (>0.2 ppm) after dT7 binding. In case of W8, the Nε1-Hε1 peak in its indole side chain showed a huge perturbation at 0.80 ppm and the R2 value of W8 significantly decreased by 8.84 s-1 after dT7 binding. K7 and N10 also showed large decreases in their R2 values after dT7 binding at 7.64 s-1 and 6.06 s-1, respectively. As 29 ACS Paragon Plus Environment

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Figure 9. Structure of the Cp-Csp-dT7 complex determined by docking calculations. The carbons in the side chains of the interacting residues K7, W8, N10, F15, F17, F28, H30, S32, Q35, K58, N63, and K64 are denoted in cyan, and those of dT7 are colored in yellow. In both molecules, nitrogen atoms and oxygen atoms are colored in blue and red, respectively. Phosphorus atoms in the phosphate groups of dT7 are denoted in orange.

expected, the micro-to-millisecond scale motions in these residues decreased markedly after dT7 stabilized the Cp-Csp structure. ΔR2eff decreased more than 9 Hz for W8, F9, N10, S11, F15, F28, V29, H30, Q35, L43, A44, V53, K58, N63, K64 and V66. Among these residues, K7, W8, N10, F15, F28, H30, and K58 are known as highly conserved binding residues, which were reported to have interactions with T-rich DNA strands.19, 40, 41 Therefore, during the simulation, the side chains of these seven residues that seemed to interact with dT7 were kept flexible. The side chains of these residues may be closely located on the surface of Cp-Csp, forming a binding interface. As shown in Fig. 9, hydrogen bonds, electrostatic, and hydrophobic interactions were monitored for the docking model. Similar to Lm-Csp-dT719, Bs-Csp-dT640, and Bc-Csp-dT641 complexes, docking model of CpCsp-dT7 showed that hydrogen bonds connected the side chain of K7 and indole Nε1 of W8 to the T2 base. The backbone N-H of N10 interacted with T1 and T3 bases. These interactions may explain the large decrease in their R2 rates upon dT7 binding. Carbonyl oxygen in the backbone of S32 in docking model formed a hydrogen bond with T3 base and the side chain 30 ACS Paragon Plus Environment

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hydroxyl group of S32 interacted with the T7 base, explaining the large chemical shift perturbation of the S32. The side chain amine of K58 in the docking model linked the sugar groups of T4 and T3, resulting in the large chemical shift perturbation and increase of hNOE. The side chain of N63 form interactions with the T5 and T6, connecting the T5 and T6 sugar groups. In the case of ionic interactions, positively charged side chain amine groups of K58 and K64 interact with negatively charged phosphate groups between T2-T3 and T6-T7, respectively. These interactions may explain their significant decrease in their ΔR2eff after dT7 binding. Aromatic rings of W8, F15, F17, F28, and H30 in the docking model formed π-π stackings with the thymidine bases, resulting in large chemical shift perturbation and decrease in the backbone motions of these residues. Overall binding interactions between Cp-Csp and dT7 were very similar to those of the Lm-Csp-dT7, Bs-Csp-dT6, and Bc-Csp-dT6 complexes as previously reported.19,

40, 41

However, there were some differences found in Cp-Csp-dT7 compared to the other Csp complex structures. F38 in loop 3 of Bs-Csp-dT640 and Bc-Csp-dT641 showed interaction with thymidine bases of dT6. However, corresponding residue, Y40 in Cp-Csp did not show any interaction with dT7. In addition, NMR experiments revealed that this unconserved Y40 in psychrophilic Cp-Csp did not show any significant chemical shift perturbation or change in backbone dynamic feature upon dT7 binding, while F38 in Lm-Csp showed large chemical shift perturbation as well as large increase of hNOE value.19 Furthermore, there were interactions between dT7 with N63 and K64 in Cp-Csp-dT7 binding model, which were not shown in the other Csp complexes. Docking model of Cp-Csp-dT7 revealed that there are extensive hydrogen bonds, electrostatic, and hydrophobic interactions between Cp-Csp and dT7. These interactions may stabilize the Cp-Csp-dT7 complex, resulting in dramatic increase in melting temperature upon dT7 binding.

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Discussion Temperature is one of the most important environmental factors influencing bacterial growth. Psychrophiles grow at temperatures approaching 0 °C. Low temperature decreases the reactivity of enzymes, transcription and translation, and membrane fluidity. It is important to understand how psychrophiles adapt to extreme low temperature. Many recent studies have reported the potential uses of microorganisms identified in polar regions,53, 54, underscoring the importance of understanding the biochemical mechanisms underlying physiological function in these organisms. Furthermore, many psychrophilic proteins retain their cold activities at low temperatures; therefore, investigating the fundamental characteristics of psychrophilic coldactive proteins is important. Under cold shock, nucleic acid secondary structures are stabilized, and bacteria express Csps to improve their inefficient transcription and translation. Although bacterial Csps are largely homologous and structurally similar, they display drastic differences in thermostability. X-ray crystallography and NMR spectroscopy have been used to determine the threedimensional structures of various bacterial Csps, including E. coli (Ec-CspA)55-57, B. subtilis (Bs-CspB)47, 58, B. caldolyticus (Bc-Csp)42, T. aquaticus (Ta-Csp)20, T. maritima (Tm-Csp)37, 48, L. monocytogenes (Lm-CspA)19, and Corynebacterium pseudotuberculosis (Cpse-Csp)59, all of which are highly conserved. All Csps have nucleic acid-binding regions (RNP 1 and RNP 2), composed of the surface of the β-barrel structure. These nucleic acid-binding interfaces primarily comprise basic residues that can interact with the negatively charged phosphate groups of nucleic acids and aromatic residues, which can interact with nucleic acid bases. In general, psychrophilic proteins tend to have flexible structures to compensate for the freezing effect in cold habitats, resulting in lower thermostability than that of their mesophilic counterparts.60 Crystal structures of several psychrophilic cold-active enzymes, including αamylase, β-lactamase, lipase, and subtilisin, showed that these proteins have fewer 32 ACS Paragon Plus Environment

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intramolecular interactions compared to their mesophilic homologs. The combined approach of experimental and computational analysis also revealed that psychrophilic DNA ligase displayed relatively high conformational flexibility and overall structural destabilization. Reductions in surface charge and increase in solvent-exposed hydrophobic surface contribute to the destabilization of the overall structure of psychrophilic DNA ligase.61 Similarly, esterases from psychrophiles have a decreased amount of hydrophobic and ionic interactions and hydrogen bonds compared to their mesophilic and thermophilic counterparts.62 These molecular adaptation strategies act in concert, making proteins act more efficiently at cold temperature. For psychrophilic bacteria, cold shock can be defined as a temperature reduction to ≤0 °C. Psychrophilic Csps also require flexible structures to allow for conformational changes necessary to facilitate the destabilization of the secondary structure of nucleic acids at low temperatures. Considering the cost, psychrophilic Cp-Csp has the lowest melting temperature in comparison with thermophilic, mesophilic, and psychrotrophic Csps (Table 1). Cp-Csp has distinctive amino acid composition, including that of basic and acidic residues. As illustrated in Table 1, the pI generally tends to decrease from thermophilic through psychrophilic Csps. Thermophilic proteins tend to be more basic than those of mesophiles and psychrophiles because of their relatively large complement of the positively charged residues Arg and Lys. Some of these positively charged residues participate in extensive electrostatic interactions or ion clusters, which contribute to the high thermostabilities of thermophilic Tm-Csp and TaCsp.20,

37

These comparisons indicate that the major differences in the Csp sequences of

thermophiles, mesophiles, and psychrophiles are the content and distribution of positively charged amino acid residues. Therefore, these features of Csps are potentially important for folding and nucleic acid binding. The ratio of basic to acidic residues is higher in thermophilic Csps than in mesophilic and 33 ACS Paragon Plus Environment

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psychrophilic Csps. In Tm-Csp, this ratio is 11/11, while that of Ec-Csp is 7/8, and that of CpCsp is very low at 6/9, since Cp-Csp has six Lys, no Arg, four Asp, and five Glu residues. A reduction in Arg residues and in the total number of positively charged residues decreases the capability of ionic interactions in psychrophilic proteins.63 Furthermore, proteins from bacteria growing in hypersaline environments have abundant acidic over basic residues.64,

65

C.

psychrerythraea 34H can be found in salty Arctic and Antarctic sea ice. The ratio of acidic to basic residues is higher in Cp-Csp than in other Csps. As shown in the sequence alignment, CpCsp is unique in having negatively charged residues such as E33 and E39 at the surface loop 3 region. The presence of a highly negatively charged surface is thought to increase solubility and maintain the function of proteins at high salinity by sequence analysis of halophilic proteins.66 Therefore, all the distinctive features of the sequence in Cp-Csp might be important in its proper function in distinctive cold and salty environments. The structural features of various Csps are summarized in Fig. 10. Previous studies have reported that the solution structures of the mesophilic Ec-Csp57 and Bs-Csp47 proteins contain two salt bridges. Thermophilic Ta-Csp not only has two salt bridges—K7−D25 and K63−E52—but also an additional ion cluster, E21−K3−E48−R66.20 Similar to thermophilic Csps, the hyperthermophilic Tm-Csp also has two salt bridges, K6−D24 and H61−E49, and an ion cluster, D20−R2−E47−K63.37 Psychrotrophic Lm-Csp also has two salt bridges, E2–R20 and K7–D25;19 however, as shown in Fig. 10, psychrophilic Cp-Csp only contains a single close-range ionic interaction, K7–D26, which is conserved among Csp homologs. Interestingly, a highly conserved salt bridge between the N-terminal domain of the β1 strand and the Cterminal domain of the β2 strand is missing in Cp-Csp. E21 at the loop 2 region in Cp-Csp does not form an ionic interaction with K3. In contrast, its corresponding partners, E21 and D20, in the ion clusters of Ta-Csp and Tm-Csp, form salt bridges with K3 and R2, respectively. Similarly, the E2–R20 salt bridge of Lm-Csp is located immediately downstream of the β2 34 ACS Paragon Plus Environment

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strand. The increase in the temperature coefficient of E21 and chemical shift perturbation of K3 upon Y51F mutation imply that non-conserved Y51 may disrupt the formation of the K3– E21 salt bridge, and the Y51F mutation may allow the formation of the K3–E21 salt bridge, thereby resulting in a 6 °C increase in melting temperature; however, further studies are required to confirm this observation. For the mesophilic Ec-Csp56, 57, the lengths of four loops (loop 1, 2, 3, and 4) are 4, 7, 16, and 6 residues, respectively. Tm-Csp37 and Ta-Csp20 have shorter loops, comprising 3, 4, 13, and 6 residues for the hyperthermophilic Tm-Csp and 4, 4, 16, and 5 for the thermophilic TaCsp, respectively. As expected, thermophilic Csps, which must endure heat denaturation, should have a high proportion of secondary structures. In contrast, the structure of psychrophilic Csps must be flexible to suitably accommodate ligand binding at freezing temperatures. The lengths of these four loops in Lm-Csp are 4, 4, 17, and 6 residues,19 while those in psychrophilic Cp-Csp are 4, 7, 17, and 6 residues, respectively. The hydrophobic cavities of both hyperthermophilic Tm-Csp37 and thermophilic Ta-Csp20 are formed by 12 residues, as are those of the mesophilic Ec-Csp57 and Bs-Csp47. The number of core residues decreases to 10 residues in psychrotrophic Lm-Csp19 and psychrophilic Cp-Csp, contributing to the decreased thermostability of these proteins. Therefore, the structural flexibility that accompanies longer surface loops and reduced hydrophobic core packing, and the number of salt bridges, are potential key factors in the low thermostability of Cp-Csp. Backbone dynamics of various Csps, including Ec-CspA56, Bs-CspB40, Lm-Csp19, and Ta-Csp20, have been investigated, and all Csps display similar dynamic charact eristics necessary for nucleic acid binding. Spin-relaxation data revealed that psychrophilic Cp-Csp has a highly flexible long loop 3 region, relative to psychrotrophic and thermophilic Csps. Upon binding to dT7, the hNOE of the residues in the nucleic acid-binding site increased drastically, and the level of conformational exchange reduced dramatically upon binding. It 35 ACS Paragon Plus Environment

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Figure 10. Comparison of the structures of Csps from bacteria adapted to environments at different temperatures. Comparison of the 3D structures of Ta-Csp20, Ec-Csp57, Bs-Csp47, LmCsp19, and Cp-Csp. The salt bridges on the protein surfaces are indicated by yellow-dashed lines. The side chains of the hydrophobic core residues are denoted in green. appears that dT7 binding somehow induces additional local stabilization in the β3 and β4 regions of Cp-Csp, compromising the highly flexible loop 3 region. Cp-Csp displayed high R2 rates mainly at the β1-β2 surface at the RNP 1 site. CPMG also confirmed the presence of a conformational exchange in this region, and additional conformational exchange in the microsecond-to-millisecond time scale appeared at the RNP 2 site. We assumed that there are chemical exchanges between the unbound and bound forms of Cp-Csp. Chemical exchange rates and the proportion of the nucleic acid-bound state within the Cp-Csp structure were evaluated using CPMG NMR relaxation dispersion data analysis. In our previous study of Ta-Csp, we found that this thermophilic Csp undergoes chemical exchanges in K7 (690 s-1), W8 (841 s-1), Y15 (616 s-1), and F17 (547 s-1), which are located at the β1-β2 surface of the nucleic acid-binding interface; the average kex value of these residues was 674 s1 20

.

Psychrophilic Cp-Csp underwent much faster chemical exchanges at K7 (3000 s-1), W8

(9695 s-1), and F15 (1350 s-1), but not at F17 (274 s-1). The average kex of the residues K7, W8, F15, and F17 in Cp-Csp was calculated as 3580 s-1, which is approximately fivefold faster than that of the thermophilic Ta-Csp.20 Therefore, psychrophilic Cp-Csp displays more rapid 36 ACS Paragon Plus Environment

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exchanges between conformers at the binding interface than thermophilic Ta-Csp, in terms of microsecond-to-millisecond time scale motions. In Cp-Csp, there are chemical exchanges with approximately 3% of Pbound occurring in both the RNP 1 (754 s-1) and RNP 2 (642 s-1) motifs. These conformational exchanges in the nucleic acid-binding surface appear crucial to accommodate nucleic acids. The three-dimensional structure and backbone dynamics data for Cp-Csp revealed a loosely packed hydrophobic core, long and flexible loop regions, and fewer ionic interactions than other Csps. The chemical exchange rates are greater and loop flexibilities are larger for psychrophilic Cp-Csp compared to other mesophilic and thermophilic Csps. In addition, the phenolic hydroxyl group of Y51 at the center of the hydrophobic packing protrudes toward the solvent, rendering the internal packing substantially less compact. Subsequently, some of the hydrophobic side chains of adjacent residues are exposed to the exterior, resulting in further reductions in hydrophobic packing. To overcome the damage caused by intracellular ice crystallization, psychrophilic proteins, such as antifreeze proteins, tend to have more surfaceexposed hydrophobic residues.67, 68 The extra hydrophobic residues on the surface of Cp-Csp, emerging through the hydrophobic core, may contribute to the protection of the protein from the damage during freezing of the surrounding solvent. Furthermore, Tyr51 may disrupt the formation of a salt bridge between Lys3 and Glu21, resulting in low thermostability of Cp-Csp. As the intramolecular interactions essential to retain the native state are reduced, psychrophilic Csps become more heat-labile than the other Csps. In contrast, thermophilic Csps functioning at high temperatures have the opposite features. In particular, ionic interactions are enhanced, primarily because of more positively charged residues, and they are adapted to maintain their folding and function at elevated temperatures.20, 37, 42, 48 The flexible structure of psychrophilic Csps, resulting from weaker intramolecular interactions (less ionic interactions, less compact hydrophobic packing, more solvent-exposed hydrophobic residues, and a lower 37 ACS Paragon Plus Environment

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proportion of secondary structures), appears to be an important factor for their molecular adaptation. Hence, the conformational flexibilities of Cp-Csp facilitate appropriate function and accommodation of nucleic acids at a lower energy for adaptations of C. psychrerythraea 34H in cold marine environments. Our findings may elucidate fundamental properties of the psychrophilic cold-active proteins and provide insights into the correlations among the structure, dynamics, thermostability, and cold shock adaptation of psychrophilic Csps.

Supporting Information Figure S1

Acknowledgement This work was supported by Konkuk University 2014. We thank Dr. Woonghee Lee for valuable help using PONDEROSA-C/S.

Accession Codes Final coordinates and NOE constraints have been deposited in the Protein Data Bank (PDB) under the accession number 5XV9 (BMRB ID: 36102).

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Tyr51: Key Determinant of the Low Thermostability of Colwellia psychrerythraea Cold Shock Protein Yeongjoon Lee, Chulhee Kwak, Ki-Woong Jeong, Prasannavenkatesh Durai, Kyoung-Seok Ryu, Eunhee Kim, Chaejoon Cheong, Hee-Chul Ahn, Hak Jun Kim, and Yangmee Kim*

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