Figure 3.4 DNA sequence o f the wild-type amiC, amiR genes.
P.aeruginosa sequence is num bered (1-1823). The E coR l and H in d lll restriction targets used to PCR clone the amiC, amlR fragment are shown in bold. A translation o f the am iC
and am iR open reading frames is shown under the DNA sequence and am ino acids are num bered on the right hand side. Primers used to sequence the coding strand (5 ’ to 3 ’) are underlined and labelled. Primers used to sequence the complementary strand (3 ’ to 5 ’) are overlined and labelled. Arrows on primers show the direction o f DNA sequencing.
980 1000
* * * * * * *
W ild type 5 ’ - CAC CTG TAC GAC ATC GAC ATC GAC GCG CCA CAG - 3 ’
His Leu Tyr Asp He Asp He Asp Ala Pro Gin
314 315 316 317 318 319 320 321 322 323 324
pRA N l111 5 ’ - CAC CTG T A C --- GAC ATC GAC GCG CCA CAG - 3 ’
F ig u re 3.5 DN A sequence alignment o f wild-type and pR A N l 111 amiC sequence showing PAC 111 mutations.
P. aeruginosa wild-type sequence is numbered (973-1005). A translation o f the am iC open reading frame is shown under the DNA sequence and amino acids are num bered. The corresponding pR A N l 111 DNA sequence alignment and amino acid m utations are shown below the wild-type sequence.
620
W ild type 5 ’ - TAC CAG GCG CGC GCC GAC GTG GTC - 3 ’
Tyr Gin Ala Arg Ala Asp Val Val
194 195 196 197 198 199 200 201
pR A N l 153 5 ’ - TAC CAG GCG TGC GCC GAC GTG GTC - 3 ’
Cys 197
B
1460* * * * *
W ild type 5 ’ - TAC GAA AGC CCC GCG GTG CTC TCG - 3 ’
Tyr Glu Ser Pro Ala Val Leu Ser
88 89 90 91 92 93 94 95
pRAN1153 5 ’ . TAC GAA AGC TCC GCG GTG CTC T C G - 3 ’
Ser 91
F ig u re 3.6 D N A sequence alignm ents o f w ild-type and pR A N l 153 a m iC and amiR
sequences showing FAC 153 mutations.
(A) P. aeruginosa wild-type sequence is num bered (613-636). A translation o f the am iC
open reading frame is shown under the DNA sequence and amino acids are numbered. The corresponding pRAN1153 DN A sequence alignment and amino acid m utations are shown below the wild-type sequence. (B) P. aeruginosa wild-type sequence is num bered (1449- 1472). A translation o f the amiR open reading frame is shown under the DN A sequence and amino acids are numbered. The corresponding pR A N l 153 DNA sequence alignm ent and amino acid mutations are shown below the wild-type sequence.
Chapter 3: RESULTS 1
B
C-Domain
N-Domain
Figure 3.7 Structure o f AmiC.
(A) Secondary structure cartoon o f AmiC. The N-domain is on the right, the C-domain on the left, a-helices are coloured red, (3-strands green and coils and loops yellow. A bound acetam ide m olecule lies between the two domains. The figure was generated using MOLSCRIPT (Kraulis, 1991). (B) Secondary structure o f AmiC. Helices are drawn as rectangles and sheets as broad arrows.
helices. The C-domain has a similar topology, with a central core of four parallel p- sheets flanked by helices, but with an additional p-hairpin anti-parallel to the edge of the central sheet. The polypeptide chain crosses over three times between the two domains, which come close together to form an extensive interface. The amide binding site lies within a pocket formed at the interface of the two domains.
Amino acid mutations from P A C lll and PAC 153 AmiC’s were modeled and analysed by comparison to the wild type AmiC structure as described in section 2.2.6.
S.3.4.2 Deletion of Asp317 and Iie318 in PAC111 AmiC mutant
The Asp317-De318 amino acid deletion is located in a surface loop in the N terminal domain on the opposite side and far from the amide binding cleft opening. In the region of the mutation, the wild type AmiC structure (Figure 3.8A) shows almost a whole three turn a-helix formed by residues 308-315, linked by a five residue surface loop to an antiparallel p-sheet. His314, present in the a-helix, forms backbone H-bonds with Asp317 and De318, which is a typical pattern seen in helices. The residues Asp317, 319 and 321 which form part of the surface loop all have their carboxylate groups (-COO") pointing out into the solvent thus creating a highly negative, hydrophilic region. In the model (Figure 3.8B), the absence of Asp317 and De318 has disrupted the H-bonding pattern with His314 causing the a-helix to loose half a turn and effectively becoming shorter. The shortening of the a-helix has not affected the hydrophobic core of AmiC since the overall positions of the helix and the p-sheet is the same, with most of the side chains in a similar conformation. The absence of Asp317 leads to the loss of one negative charge from the surface loop causing the region to become less negative.
It is possible that the negatively charged, frve residue surface loop described above interacts with a positively charged region on the AmiR protein. In which case loss of negative charge in the P A C lll (constitutive but sensitive to butyramide repression) AmiC mutant would weaken the interaction between AmiC and AmiR thus providing an explanation for the constitutive phenotype of P A C lll. In the presence of butyramide the overall AmiC structure would be slightly modified so as to
Chapter 3: RESULTS 1
B
He 318 Asp 321 Asp 319 Asp 319 Asp 317 His 314 Asp 317 His 314Figure 3.8 Structure o f AmiC residues 308-335 (wild-type) and 308-333 (PAC 111).
(A) Wild-type AmiC (308-335). Backbone is shown in green and residues are coloured by type: His (purple), Asp (blue), He (red). (B) PACl 11 AmiC (308-333). Backbone is shown in red and residues are coloured by type: His (purple) and Asp (blue). Figures were generated using INSIGHT II (Biosym/MSI, San Diego, CA, USA).
accommodate for the loss of negative charge and would retain the wild-type degree of interaction with AmiR.
3.3.4.3 Substitution of Arg197 to Cys197 AmiC mutation in PAC153
The Arg to Cys substitution at position 197 is located on a surface loop in the C- terminal domain far from the AmiC amide binding site. In the region of the mutation, the wild type AmiC structure (Figure 3.9A) shows that the ^-strand formed by residues 170-177 is parallel to the a-helix formed by residues 183-196 and with a number of hydrophobic contacts between them. In the wild type, Arg 197 is the first loop residue after the a-helix (183-196). Its side chain is solvent exposed with the guanadinium group (-N H -CN H2^-NH2) forming an electrostatic interaction with the backbone
carbonyl group of Tyrl94. From the model, it is evident that Cys 197 (Figure 3.9B) also appears to be solvent exposed. Backbone H-bonds to lie 193 are conserved in the mutant as in the wild-type causing no change in tertiary structure in this region. There is loss of a surface positive charge due to the absence of Arg.
In view of these results the phenotype of PAC 153 (formamide inducible) cannot be directly attributed to the Arg to Cys substitution found in AmiC. There appears to be no structural change in the backbone compared to the wild-type with the loss of a positive charge being the only difference.
The next stage of the investigation was to overproduce AmiC and AmiR and see whether they formed a complex.