(4) X!"#$%&'()*+,-./01β-lactamase2Cs+34-567829:!"# $%&'( $)*+, $-./0 $1234 $5678 $9:;<4 $56=> $?@A3
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122

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ÜÝ%/â¶·ãä@&åDEæÀ( !"#$OM@çèäC/éêO¾@ëÔtìHLí]?HT
m\b/Asn-Gly ÜÝ@ Gln-Gly ÜÝ.îïtì,ðñÚ&Û HaBLAgÓÔ/NQ-HaBLAh@òóØô]C,Tõ NQ-HaBLA (ËÌM@Íö÷ø pET-15b ù#
ú(Novagen }Ð)@ ÐCD/E. coli BL21 Star
(DE3)(ËÌMÍÎ@û8/N üýþ. His-tag @ÿ C, NQ-HaBLA @ tì,!/Ni-IMAC "#fgPhyNexus }Ðh@8D NQ-HaBLA ($%Ð@ûu,T&8D/NQ-HaBLA ( His-tag @thrombin .p'()C/Resource Q "#fgGEHealthcare }Ðh@8,*23!+Î#,-./#01ú.p'23%Ð'@4,T!"#$%&'(&)*+(,-./0123!
(456(78923!:;(<=>?78@AB
CDEFGH?I/JK?LMNOMPAQR@SGHTUV( !"#$WXYZ[(01@EFGH\]%^V_`aD8HTC>C/ !"#$WXYZ[b%?8cdefgCsh@ijCDEFCkH(>/l,/m(EFno%I(pk?Rq@CD8H(>r%s`>.taD8?>u,Tv./wx4yz{|}~LMy4 .puDta,Cs(&'(@_H,-9/ !"#$@
C,Cs@GH,-.%/ !"#$.
HCs (EF(C9Gt9EFno(Rq@sGH\]W]`a,Tm\b%/ !"#$]Cs ( (¡¢.£C,T !"#$%/¤N¥N¦§¨N©§¨?I(ª«¬­®.&¯GH°±W H !"#$b²HT !"#$%/ª«¬­®.³´GH,-.UVµ¶·¸µ@¹SC/UV(º45@SGH\]>`/Cs @¹»¼?0123!@EFGH½¾W²H]
%,T¿¡¢b%/ !"#$(ÀbÁÂÃÄª8µ¶·¸µ¹Å@SC[(Asp + Glu) / (Arg+ Lys) = 2.11]gÆ1h/ÇÈ/ÉÊ±(ËÌMÍÎ.puDÉÅÏÐW½¾?±Chromohalobacter sp. 560ÑÒβ-lactamase gÓÔ/HaBLAh.È8D/ÕÖRq(s]Cs EFno(×@ØÙ,TI(;< !@8Dûu,T&8D/EæW"
C,#$ÿ%.8D/ !"#$«¬/;<«¬r@2³äC,T23Ä.%/&¬ 20 C/ !"#$«¬ 30 mg/mL/200 mM 'µ"(defàá18%(w/v) PEG8000 @¹» 100 mM MES buffer(pH6.5)(Eæä !gÓÔ/#$ Ah>`/éê89.³b)H NQ-HaBLA Eæ@4,T

2. UV
2-1 WXYZ
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2-3 ^_K`ab$cd$X efghijb
l=/NQ-HaBLA(ÕÖRq@sGH,-./ª*+(,ú -.¡¢QR(PF-AR./0ta,NW12Ag121.000 Åh@CD/#$A.8D++++2-2 [\]Z
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!"#NQ-HaBLA$%&X $%'()*+,#./01$%'(2&Cs )3456#781Cs)9:$%;!"8NQ-HaBLA $%)#$,1
SAGA-LS&BL07)</0X =%>&'(?@XAB'()CD#-E&'(FG1)*1.000 Å&X)</0AB+HIJ,K)-.,#L1/M&$%)</01Cs&X =%>&01NO"6)*
2.166 Å&X 8PQAB+HIJ,K)-.,#20&+HIJ,KG1NVHRS3THampton
ResearchUVW)45$XY,#100 K&Z678[8\/0-.,#-AB+HI&9]^_?@:;G
XDS 1>`<a=8P6>?bc@(GMOLREP)</0CD#-d#1'(ef;GREFMAC5?@Coot )</0CD#-X +HI-.?@'(ef;&ABC)g28D5++1)

2)

3)

4)

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Cs )$,0/#TV2aW-Cs YTrp189l&
vmv&q`TCδ21Cε21Cε31Cζ21Cζ31Cη2WY&r&stG3.2u4.0 ÅF¡D#-d#1Cs
YGln186?@Thr188pl&`Y&r&stG
3.1u3.7 ÅF¡D#-Cs – Orst&vwCG3.1u
3.3 ÅF¡Q 1E&CYTxMy,0/6zAk,#Cs ${bG1Cs &{¢Q8Na £Ca )9:]"78[T78AWFG|>`)$,0/#TV2bW-d#1E&Cs ${b&'(G1$%;!"&78)¤¥80TY|};¦§1}~78AuD£ SRv)Mg 1Ca 1
Sr 8#8\/01Gln186YThr188&pl`b<?@Trp189l&RMSDG0.18 Å¨[F¡D#-fD01Cs 8,#${b&'(N]©&ª«)_8yy1Cs ¨&SRv¬­)q56 SRv8 ,0pNZ/EYN1m/Cs p&®¯8sD0/6Y°!"6++++5)+++2++2+2+2+!"#$
PF-AR&NW12A8\/0hE8CD#78A&X$%*F&$11NQ-HaBLAGG%ijkl&
class-c β-lactamaseYm/no'(c/p)q56E
YN+!r8sD#TCα&RMSDG1.3 ÅHtW-M
I1>`gJ&KuLMvwN3GO&β-lactamase
YxPy=sQ1zp{bTSer641Lys671Tyr1501
Glu2721Lys315W&QR)S/01TY|}N~uFU7!"0/#TV1W-~uft1O&β-lactamaseY,0WX8m/C)D,#Tg1WE"!&Yr!1NQ-HaBLA&>`gJG S
1! β-lactamase <IJK"LMNOP4Q9'(a)Rv&$856EYNDZ"#HaBLA(b) R S T " U ; V W Enterobacter
EFSAGA-LS&BL07FG1Na / Cs &t) aerogenes/XY
EaBLA'Z6[M\/16]M\'
0/100 mMT78BW175/25 mMT78CW190/10 mM
T78DWY#3[\&$%;]"8NQ-HaBLA$%)#$,0X $%*F)^,1Cs&X =%>&01)<,01>`gJ8$,#Cs &34)CD#-&$11=%>&_H`V8\/01
78BFG17.5σa3178CFG17.0σa317
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2! HaBLA " Cs #$%&'(a) Cs ()*+,-B./(b)Cs 0()*+,- A.'1"23456 5 789${bNhi,1&{bGNa NCs &9j9d ":;<=>?@ABC3D'E"23456/2 7"6]"FCs ),0$56EYN+!r8 89" 2F - F FG3HC3D'
3.

+++++++++++++o䋭㪈㪏䋭c+!"!"#$%&NQ-HaBLA #'&Cs #()*+,-./ Na 0 Cs 1 9 2345/!"67 Cs ,89:;<=./>?0#$./@A0BCD#EF)G%HIB:) Cs 89J<=>?1KL&',MN./@A6&Cs #(./O?PQ,)R:;
NQ-HaBLA 1 Cs *+*S89*,,#-../E
T1UVWX+YP/00Z[#E/G4)&11U
VWX+# Cs <=>?,23K4./@A7Z[#E/G@5C1\5,6;&%]' Cs 78UVWX+9^1_`,a:.G !";<'& Acta
Crystallographica Section D #=>b5) G[5] Haynes, W.M. et al., “CRC Handbook ofChemistry and Physics, 94th ed.”, Boca Raton: CRCPress, pp. 12-11–12-12 (2013).
[6] Arai, S. et al., “Structure of a highly acidic
β-lactamase from the moderate halophile
Chromohalobacter sp. 560 and the discovery of aCs+-selective binding site.” Acta Crystallogr. D, 71,541-554 (2015).

#$%&45
1$678Asp + Glu&
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1$67*94.

++++++++! "#$+1%&'()*+,-./01(23β-lactamase (HaBLA)

β-lactamase (EaBLA)

6)[1] Kabsch, W. “XDS”, Acta Crystallogr. D, 66,125-132 (2010).
[2] Vagin, A. & Teplyakov, A. “Molecularreplacement with MOLREP.” Acta Crystallogr. D,
66, 22–25 (2010).
[3] Murshudov, G.N., et al., “REFMAC5 for therefinement of macromolecular crystal structures.”,
Acta Crystallogr. D, 67, 355–367 (2011).
[4] Emsley, P. & Cowtan, K. “Coot: model-building
tools for molecular graphics.”, Acta Crystallogr. D,60, 2126–2132 (2004).

! DE2./

1.000 Å( FGHIJX

ANQ-HaBLA

B

18 + 913 + 18

2.11

0.94

-

1.3

*#/"+#,+-&:;(<!=>?@AB< !=CRMSDe

C

1.49-2( FKLMN(OPQX

2 Cs 2+10 Ca

1 Cs 2+
10 Ca

Space groupP31
a = b = 114.9
c = 67.6α =β = 902+

12 + 172 Cs 2+
10 Ca

0.1 M Cs+
0.2 M Ca2+

11 Ca

32 + 25!"#$%&

P31
a = b = 115.4
c = 67.7α =β = 90
γ = 120
2.00(2.05-2.00)
3.7 (4.0)
8.1 (31.2)
97.7 (99.9)
15.3 (12.4)
19.0 (21.9)
22.4 (26.0)

0.1 M Na+
0.2 M Ca2+

Metal ions in an
asymmetric unit

Enterobacteraerogenes

D

Metal ion in themother liquor

Redundancy
Rmerge (%)
Completeness (%)
I /sigI (%)
R-factor (%)
Rfree (%)

sp.560

3WS210 mM Cs+
90 mM Na+
0.2 M Ca2+
P31
a = b = 115.3
c = 67.5α =β = 90
γ = 120
2.10(2.15-2.10)
3.2 (2.4)
7.9 (40.9)
93.7 (91.5)
8.7 (1.9)
14.4 (12.5)
17.4 (17.0)

3WS0

Resolution (Å)

Chromohalobacter

C3WRZ

γ = 1201.80(1.85-1.80)
5.4 (4.8)
8.3 (26.3)
99.9 (99.4)
23.1 (15.5)
17.2 (15.3)
22.1 (19.8)

EaBLA

3WS1
25 mM Cs+
75 mM Na+
0.2 M Ca2+
P31
a = b = 115.1
c = 67.3
α =β = 90
γ = 1201.80(1.85-1.80)
5.7 (5.6)
4.1 (30.8)
99.2 (98.0)
18.4 (3.8)
18.5 (20.4)
21.1 (22.8)

PDB ID

Unit cell (Å, º)

HaBLA++䋭㪈㪐䋭+

10-3'()$%&

C-0.86

10-3