of the Type II Acyl Carrier Protein (ACP) Utilizes a âUbiquitin

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The 'recognition helix' of the type II Acyl Carrier Protein (ACP) utilizes a 'ubiquitin interacting motif (UIM)' like surface to bind its partners Usha Yadav, Richa Arya, Suman Kundu, and Monica Sundd Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00220 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Biochemistry

The 'recognition helix' of the type II Acyl Carrier Protein (ACP) utilizes a 'ubiquitin interacting motif (UIM)' like surface to bind its partners

Usha Yadav‡, Richa Arya†, Suman Kundu† and Monica Sundd‡ ‡

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.

†

Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India



To whom correspondence should be addressed: Monica Sundd, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110 067, India, Tel. +91-11-26703823, +91 11 26162125 Email:[email protected].

Running title: Recognition helix of type II acyl carrier protein utilizes a UIM motif like surface

1 ACS Paragon Plus Environment

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Abbreviations used: ACP, acyl carrier protein; FAS, fatty acid synthesis; UIM, ubiquitin interacting motif

Keywords: acyl carrier protein; fatty acid biosynthesis; ACP; ubiquitin; UIM; NMR


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Biochemistry

ABSTRACT Interaction interfaces comprise a finite number of sequence/structural motifs, that are often repeated in nature. Here we show how a helical motif present in the acyl carrier protein, involved in multi-protein interactions, displays similar binding interface as the "ubiquitin interacting motif (UIM)". Analysis of the crystal structures of the ACP-enzyme complexes gave the first hint, that the helix II of the acyl carrier protein ('universal recognition helix'), utilizes UIM like non-covalent interactions to associate with the type II fatty acid biosynthesis (FAS) pathway enzymes. The ACP interacting functional surface of the FAS enzymes comprises positively charged residues, flanking a central hydrophobic patch, akin to ubiquitin. Our NMR chemical shift perturbation studies, relaxation studies, and SPR measurements, unequivocally show that the helix II of ACP behaves like a UIM motif in its interactions with ubiquitin, by binding the Ile 44 functional surface of the latter. A synthetic peptide with the ACP helix II sequence showed equivalent binding to ubiquitin. The evolution of similar interaction motifs in the two systems has probably been driven by functional constraints, as both ACP and UIM participate in multi-protein interactions.


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Introduction The type II fatty acid biosynthetic machinery has been well characterized in E. coli, and its proteins serve as a paradigm to understand the interactions of the catalytic enzymes of the pathway. Acyl carrier protein is an indispensable component of the pathway, as it sequesters the growing acyl chain in its hydrophobic core, and shuttles it to the active site of the catalytic enzymes upon interaction. In its lifetime, it associates and dissociates with innumerable proteins/enzymes. ACP has been structurally characterized in E. coli, S. oleracea, P. falciparum, H. pylori, L. major1-4 etc. Apart from fatty acid biosynthesis, ACP plays a pivot role in the synthesis of polyketides,5,

6

oligosaccharides,7 biotin, nonribosomal peptides,8,

9

lipid A,10 and quorum sensing compounds.11 Acyl carrier protein is one of the most abundant proteins in the cell. All type II ACPs comprise a four helix bundle, enclosing a hydrophobic cleft, that protects the prosthetic group, as well as the growing acyl chain. It's interaction with different pathway enzymes is mediated by an amphipathic helix, also known as the "universal recognition helix", owing to its ability to bind a large number of binding partners.12 Sequence comparison of the type II ACP's suggest that the residues important for interaction are conserved. That's probably why, ACP from distant organisms viz. E. coli and Rhizobium can be swapped, without any major loss of function.13 Notably, the FAS enzymes, that associate with ACP, differ remarkably in their sequence and structure. Yet an ACP recognition motif is conserved throughout near their active site. Comparison of the ACP-FAS structures suggest, that the the helix II of ACP interacts with its partners in a manner comparable to the ubiquitin interacting motif (UIM), and the noncovalent interactions of the FAS enzymes are analogous to ubiquitin. In ubiquitin,


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Biochemistry

the positively charged residues, Lys 6, Lys 11, Arg 42, His 68 and Arg 74, in conjunction with the patch of hydrophobic residues they enclose, Leu 8, Ile 13, Ile 44, Gly 47, and Val 70, interact with the hydrophobic and negatively charged residues of UIM.14 Mutation of any of the UIM interacting residues of ubiquitin reduces binding, i.e. substitution of His 68, Gly 47 or Ile 44 with an Ala eliminates binding of ubiquitin to the Vps27 UIM in pull down assays.15

The canonical UIM motif in contrast is a ~20 amino acid long amphipathic helix, with the consensus sequence; Ac-Ac-Ac--x-x-Ala--x-x-Ser-x-x-Ac, where  is a hydrophobic residue (Leu/Ile/Phe/Tyr), and 'Ac' an acidic residue. It is present in most ubiquitin-interacting proteins,16 involved in endosomal sorting and lysosomal trafficking.17 By virtue of this motif, endosomal proteins recognize and associate with the ubiquitylated proteins. Mutation of the conserved residues of UIM reduces ubiquitin binding. For instance, in the Hrs UIM (Hepatocyte growth factor regulated tyrosine kinase), mutation of the acidic residues 259EEEE262 at the N-terminus to 259AAAA262 eliminates Hrs binding to ubiquitin completely, while the mutation of the conserved Ala reduces binding ten-fold. Mutation of E273 (present at the C-terminus) reduces binding 2.5-fold, and Ser to an Ala mutation reduces 1.5-fold.17 Similarly, in Mus musculus Eps15 UIM, mutation of the three acidic residues E879, E881 and D882 to an Ala results in decreased ubiquitination,18 while in HsEPSIN1, mutation of 183EEE185 to Ala-Ala-Ala, impairs binding to VEGFR2.19

Interestingly, the UIM motif, and the helix II of ACP, both function as recognition motifs, engaging in diverse protein-protein interactions. Therefore, the conserved nature of interactions in the two proteins may be evolutionarily governed by their function. In this study,


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we demonstrate that the helix II of ACP and UIM are quite similar in structure, sequence and interactions, providing explanation for the unique ability of ACP helix II to associate with multiple binding partners. Our studies show that ACP helix II can bind the Ile 44 functional surface of ubiquitin (known to bind UIM), followed using various biophysical techniques.

EXPERIMENTAL PROCEDURES

Cloning, expression and purification The E. coli ACP gene was cloned in a TA cloning vector, followed by subcloning in a pET28a vector. ACP was expressed upon induction with 1mM IPTG. Cells were harvested and the lysate was sonicated, followed by Ni2+-NTA chromatography. The bound protein was eluted with 50-200 mM Imidazole. The N-terminal His tag was removed by thrombin cleavage using immobilized thrombin. Human ubiquitin was expressed and purified as described.20 Uniformly labeled [1H, 15N, 13C] proteins were prepared by growing E. coli transformed with the desired vector, in M9 media, containing

15

N NH4Cl (1g/l) and

13

C glucose (2g/l) (Sigma,

U.S.A.).

NMR data acquisition NMR samples comprised uniformly labeled [1H,

15

N,

13

C] protein, in 50 mM sodium

phosphate buffer, pH 6.0, 0.5% sodium azide, 90% H2O, and 10% D2O. Two and three dimensional NMR experiments, viz. 1H15N HSQC, HNCACB, CBCAcoNH, etc. were acquired on a Bruker Avance III 700 MHz spectrometer, equipped with a triple resonance probe (QCI/TXI), installed at the National Institute of Immunology, New Delhi, India. NMR data were processed on a workstation running Red Hat Enterprize Linux 5.0, using NMRPipe/NMRDraw21 and analyzed using Sparky.22 The data were multiplied by a phase 6 ACS Paragon Plus Environment

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Biochemistry

shifted sinebell apodization function in all dimensions.

15

N13C spectra were referenced

indirectly using Sodium 2, 2-dimethyl-2-silapentane-5-sulfonate (DSS) as a chemical shift standard.23 1H15N HSQC spectra were acquired using 1024 data points (t2 dimension) and 512 data points (t1 dimension). The data were linear predicted in the forward direction for up to half the number of experimental points in the indirect dimensions. Constant-time 15N CPMG relaxation dispersion experiments were acquired at 285 K on a 700 MHz NMR spectrometer, using pulse sequences as described.24 A Trelax value of 40 ms was used for the experiments. A reference spectrum was acquired, without a constant-time CPMG element, along with 10 spectra with varying CPMG frequencies viz. 25, 50, 75, 100, 150, 200, 250, 300, 400, 500 and 600 Hz in duplicates. Sixteen scans per FID were recorded with a relaxation delay of 2.0 s. The pseudo-3D data were processed using NMRPipe,21 and the peak intensities were measured using Sparky.22

Saturation transfer difference experiments were acquired on a sample containing ubiquitin-ACP helix II peptide in 1:8 molar ratio, using the Bruker pulse sequence. Control experiments were performed on a ubiquitin sample without the peptide. The ligand peaks observed in the 1D STD experiment were assigned based on 1H-TOCSY and 1H NOESY spectra, using a mixing time of 80 and 200 ms., respectively.

To determine the binding strength of the interaction of ubiquitin with E. coli ACP/helix II peptide, and AcpS (Acyl carrier protein synthase) with UIM, Autolab ESPRIT Surface Plasmon Resonance (SPR) spectrometer was used. N-hydroxysuccinimide (NHS, 0.05 M)/N-ethyl-N-(diethylaminopropyl) and carbodiimide (0.2 M) were used to first activate


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the gold surface. Ubiquitin (20 µM) in 20 mM sodium phosphate buffer, pH 6.0 was immobilized on the two channels on the gold surface, test (analyte), and control. After immobilization, the surface was blocked with 100 mM ethanolamine and regenerat ed using 50 mM NaOH. In case of AcpS, 20mM sodium phosphate buffer, pH 7.8, and 100mM NaCl was used for immobilization and running. The association kinetics were monitored for 300s, followed by the dissociation kinetics for 150s. Increasing concentration of E. coli ACP/helix II peptide or UIM in running buffer) were injected on the sensor surface. SPR signals from the control panel were subtracted from those resulting from the binding of E. coli ACP/helix II peptide to ubiquitin or UIM to AcpS, using an in-line reference signal, and the subsequent sensorgram were analyzed. All experiments were recorded at room temperature (298 K), and analysed by Autolab SPR Kinetic Evaluation software.

NMR Data Analysis Chemical Shift perturbations Changes in HN have been reported as average chemical shifts (HNAve) derived from the equation25 HNAve = [(HN)2 + (N/5)2]1/2

------- [1]

where HN is the change in chemical shift in the proton dimension, and N is the change in the nitrogen dimension. One standard deviation has been used as a cut off for significant chemical shift change, and is represented as a discontinuous horizontal line in the figures. Relaxation dispersion experiments R2eff was extracted from a series of CPMG constant-time relaxation dispersion experiments using NESSY26 according to the equation:


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Biochemistry

R2eff = 1/TCPMG ln I(0)/I(CPMG) Where TCPMG is the constant CPMG time, I(0) is the intensity of the peak in the reference spectrum and I(CPMG) is the intensity of the peak at the CPMG frequency (CPMG). The data were analyzed as described previously.27 RESULTS The Type II Fatty acid synthesis (FAS) pathway enzymes display a conserved ubiquitin like "recognition motif" Analysis of the superimposed structures of ACP in their complexes with the FAS enzymes suggest that the ACP binding surface of the enzymes is quite similar to the ubiquitin functional surface, i.e. both comprise a central patch of hydrophobic residues, flanked by positively charged residues. Figure 1A shows the molecular surface of ubiquitin (colored based on coulombic charge), in complex with the Hrs UIM motif (colored yellow). Figures 1(B-F) display the complex of the acyl carrier protein (ACP) with the enzymes of the fatty acid biosynthesis pathway (FAS). In the structures, helix II of ACP is shown as ribbon (colored yellow), and the enzyme surface is colored based on coulombic charge. Figure 2A shows the FAS enzymes in the PDB 4DXE, 4ETW, 4IHF, 4JZB, 4KEH, 3EJD, 3NY7, 2FHS, 2XZ0 and 1F80, overlaid together, backbone shown as pink ribbon, positively charged residues as sticks (colored by heteroatom), and the backbone of ACP interacting hydrophobic residues as yellow ribbons. The figures were prepared by overlaying the ACP molecules in their complexes with the FAS enzymes, using the matchmaker option of Chimera.28

Helix II of E. coli ACP displays a UIM motif like surface


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Conversely, the helix II of ACP also displays striking similarities to the UIM motif. Figure 2B shows the helix II of E. coli ACP (colored magenta, PDB 1T8K), docked on the UIM in the ubiquitin-UIM complex, PDB 2D3G (colored cyan), using the matchmaker option of Chimera. Ubiquitin interacting side chains of UIM, and the corresponding residues of ACP helix II are shown as sticks (colored by heteroatom). Surprisingly, in most type II ACP's, the helix II is conserved. Amino acid sequence of the UIM of some of the ubiquitin binding proteins has been compared (Figure 3A) with the ACP helix II sequence (Figure 3B). In the UIM motif, XXA sequence is conserved, akin to the XXV sequence in the type II helix of ACP. Valine 40, Val 43 and Met 44 of the ACP "recognition helix" are analogous to Leu 263, Ala 266, and Leu 267 of the Hrs UIM, as illustrated Figure 3. In both the proteins, this conserved sequence is flanked by carboxylates at the N- and C-terminus (Figure 3 A&B). Asp 35 and Asp 38 of ACP helix II reside in a pattern similar to 259EEEE262 of Hrs UIM. Glutamate 47 and 48 of ACP helix II are analogous to Glu 275, and Glu 276 of the Hrs UIM, as shown in Figure 2B. Notably, all the aforementioned residues of ACP i.e. Asp 38, Val 40, Glu 41, Val 43, Met 44, Glu 47, and Glu 48 are crucial for its interaction with the FAS enzymes, as illustrated in Table-1.

Loop I and helix II of ACP display significant chemical shift perturbations upon ubiquitin interaction Owing to the sequence and structural similarity between ACP helix II and UIM, binding of apo-ACP to ubiquitin was followed in solution using NMR. 1H15N labeled apoACP was titrated with increasing concentration of unlabeled ubiquitin, till 1:3 ACP:ubiquitin ratio was achieved. Significant changes in chemical shift were observed for Gly 12, Glu 30,


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Biochemistry

Asp 31, Leu 32, Asp 38, Glu 41, Leu 42, Val 43, Ala 45, Leu 46, Glu 47, Ile 54, Glu 57, and Ile 62, as shown in Figure 4A. Majority of these residues map to loop I and helix II of ACP, illustrated in Figure 4B. Ile 54 HN displays relatively large chemical shift changes, probably due to the lengthening of the hydrogen bond between Glu 47 side chain and Ile 54 amide, a feature common to most type II ACP's.4 The aforementioned residues of helix II are essential for ACP function, playing an important role in its interaction with FAS enzymes, as revealed from the X-ray structures (Table-1). Similarly, NMR studies have also shown the involvement of these residues in the interaction of ACP with AcpS,29 β-ketoacyl-acyl carrier protein reductase (FabG),30 and 3hydroxyacyl-ACP dehydratase (FabA)31.

Chemical shift perturbations suggest that ubiquitin binds ACP in a manner analogous to the ubiquitin interacting motif (UIM) To discern which residues of ubiquitin associate with ACP, 1H15N ubiquitin was titrated with increasing concentration of unlabeled E. coli apo-ACP, till a molar ratio of 1:3 was attained. Figure 5A shows the changes in the average backbone amide chemical shifts of ubiquitin (determined using equation-1) upon titration with E. coli ACP. Valine 5, Thr 7, Ile 13, Thr 14, Asp 32, Glu 34, Leu 43, Gly 47, Lys 48, Gln 49, Leu 50, Leu 69, Val 70, Leu 71, Arg 72, Leu 73, Arg 74, and Gly 75 of ubiquitin display significant chemical shift change (>1SD). In the ubiquitin-UIM interaction, Ser present at the C-terminus of UIM participates in a side chain to backbone hydrogen bond with Gly 47 amide of ubiquitin.17 In the helix II peptide, Ser is present at the N-terminus instead, i+4 residues away from Val 40 of ACP. Mutation of Ser


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36 of ACP to an Ala did not cause any change in the magnitude or pattern of chemical shift perturbation of 1H15N ubiquitin (data not shown), suggesting that Ser 36 does not participate in any hydrogen bond interaction.

Chemical shift perturbations suggest equivalent binding of ACP and the helix II peptide A synthetic peptide with the sequence 'Acetyl-GADSLDTVELVMALEEE-NH2', corresponding to residues 33-49 of E. coli ACP, comprising loop I & helix II was synthesized. 15

N13C labeled ubiquitin was titrated with the peptide, attaining a final 1:8 molar ratio.

Noticeable chemical shift changes were observed in the amides of Val 5, Thr 7, Ile 13, Thr 14, Ile 23, Asn 25, Asp 32, Glu 34, Gln 41-Ile 44, Gly 47-Glu 51, Ile 61, His 68-Gly 75, as illustrated in Figure 5B. The magnitude of chemical shift changes observed in the ubiquitin backbone upon helix II peptide binding were nearly equal to those observed upon full length ACP titration (Figure 5A & B). Interestingly, the aforementioned residues of ubiquitin form the Ile 44 functional surface, that is known to bind Stam1 UIM, as shown in Figure 5 C. A region of the 1H15N HSQC spectra of ubiquitin (Figure 5D), displaying some of the amide resonances that experience chemical shift change upon helix II peptide titration. In the figure, with increasing concentration of the helix II peptide, a systematic change in chemical shift was observed. Figure 5E displays the changes in the Ile 44 C1 resonance in the 1H13C HSQC spectra of ubiquitin free (red), and complexed to the helix II peptide (blue). Figure 5F and 5G compare the surface residues of ubiquitin that display significant (>1SD) changes in their backbone amide chemical shift (colored red) upon interaction with the helix II peptide and Stam1 UIM, respectively.


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Biochemistry

Ubiquitin binds (apo-, holo-, C8-) E. coli ACP, as well as other type II ACPs via the same functional surface As holo-/ acyl-ACP are the natural substrates of FAS enzymes, we followed their interaction with ubiquitin using NMR. As illustrated in the multiple overlaid 1H

15

N HSQC

spectra (Figure 6), peaks for unbound ubiquitin (red peaks), ubiquitin bound to E. coli apoACP (orange), holo-ACP (black) and C8-ACP (cyan) are shown. Perturbations were of slightly larger magnitude upon binding holo- or C8- ACP, compared to apo-ACP. A similar pattern of chemical shift change was observed upon addition of P. falciparum apo-ACP (green peaks), and L. major (blue peaks) apo-ACP, though the perturbations were relatively smaller even at 4 fold molar excess of ACP. The spectra of ubiquitin complexed to various ACP have been compared with the spectra of ubiquitin-Stam1 UIM complex (magenta peaks), present in 1:6 molar ratio. Interestingly, the ubiquitin residues displaying chemical shift perturbations were invariably the same in all the binding studies, whether the ligand was ACP or UIM.

Relaxation Dispersion measurements suggest ms. motions in the backbone of ubiquitin complexed to the helix II peptide Previous studies have shown that the binding of UIM to ubiquitin induces slow motions in the ubiquitin backbone.20 Thus, relaxation dispersion measurements were carried out on free ubiquitin, as well as ubiquitin complexed to the ACP helix II peptide, at 285 K to understand whether the helix II peptide induces similar dynamics (Figure 7). In the free ubiquitin, the average R2 values were ~33 sec-1, and in ubiquitin complexed to the helix II peptide, the values were relatively higher ~38 sec-1. Slow motions arising due to Ile 23, Asn 25, and Asp 52 were not detectable at 285 K in the free ubiquitin sample. Hence no residue


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required Rex term in the free ubiquitin at 285 K, shown as empty circles (Figure 7A). However, in the sample comprising ubiquitin complexed to the helix II peptide, several residues required Rex term for data fitting to a two-state slow exchange process using Carver Richards equation32 shown as filled circles in Figure 7A, similar to the motions observed upon UIM interaction.20 Figures 7B & 7C show the relaxation dispersion profiles for some of residues viz. K48, L69 and R72 in the free ubiquitin, and in the ubiquitin sample complexed to the helix II peptide, respectively. The above mentioned residues required Rex term for data fitting in the sample complexed to the ACP helix II peptide.

Interaction of ubiquitin with E. coli ACP and helix II peptide using SPR To determine the binding affinity of ubiquitin for E. coli ACP, and the helix II peptide, SPR measurements were carried out by immobilizing ubiquitin on the surface of a sensor chip, and passing increasing concentrations of E. coli ACP (1, 6.4, 10, 20, 40, and 80 µM) over the chip surface. Figure 8A shows the SPR sensorgram, and the binding curve for the ubiquitin-ACP interaction is shown in inset. The two proteins interact in the micromolar range, with a calculated affinity constant (KD) of 35.3  5 μM. Figure 8B shows the binding of ubiquitin to the helix II peptide. Binding was weaker, and a KD of 700 ± 50 μM was observed. As the helix II peptide binds ubiquitin in a manner analogous to UIM, we asked whether the interaction occurs vice versa. Thus, E. coli AcpS (a representative of FAS enzymes) was immobilised on the sensor chip surface, and increasing concentrations of Hrs UIM was passed over it. As illustrated in Figure 8C, weak binding was observed between E. coli AcpS and Hrs UIM, with a KD of 600 ± 22 μM. Though no structural data is available for this interaction, based


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Biochemistry

on PDB 1F80, and 5VCB, we speculate that UIM would bind the same positively charged patch, present between two subunits in the trimeric AcpS, where the ACP binds.29

Saturation Transfer Difference experiments STD measurements were carried out to identify the peptide resonances that interact with ubiquitin, using 1:8 ubiquitin:helix II peptide ratio. Peptide resonances were assigned by superimposing the two dimensional 1H-TOCSY and 1H-NOESY spectra, and sequentially walking along the back bone in the NH-NH and NH-H region of the spectrum, as shown in Figure 9A. This allowed us to identify and assign all the observable resonances. The peaks in the 1D STD spectra of the ubiquitin-peptide complex were subsequently assigned. As shown in Figure 9B, NOEs were observed for the Hβ and H protons of Val 8 and Val 11 of the helix II peptide (Acetyl-GADSLDTVELVMALEEE-NH2), corresponding to Val 40 and Val 43 of E. coli ACP.

DISCUSSION Protein functional surfaces encompass only a small subset of the total residues of a protein, that perform specific function.33 Alanine scan suggests that only 16 out of 64 surface residues of ubiquitin are necessary for its vegetative growth in yeast.34 Though the list of binding partners is >1000, only four distinct functional surfaces have been identified so far in ubiquitin, formed by 1SD upon F) ACP helix II peptide binding, and G) Stam1 peptide interaction. Figure 6. Ubiquitin binds apo-, holo-, C8- E. coli ACP, and other type II ACPs in a manner analogous to Stam1 UIM. Multiple overlaid 1H15N HSQC spectra of free ubiquitin, and ubiquitin


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Biochemistry

upon binding apo-, holo-, C8- E. coli ACP, apo-L. major ACP, apo- P. falciparum ACP, and Stam1 UIM. A ubiquitin-ACP ratio of 1:3 was used in E. coli ACP samples, while a ratio of 1:4 was used for P. falciparum and L. major ACP. Ubiquitin:Stam1 molar ratio was 1:6. The color coding for each of the binding interaction is shown in the figure. Figure 7.

15

N Relaxation Dispersion measurements. A) Rex as a function of residue number for

free ubiquitin (empty circles), and ubiquitin in complex with the ACP helix II peptide (filled circles). 15N constant time relaxation dispersion profile for Lys 48, Leu 69, and Arg 72, in the B) free ubiquitin, C) ubiquitin complexed to the ACP helix II peptide. Figure 8. Surface Plasmon Resonance measurements of ubiquitin-ACP interaction. SPR sensorgrams for the binding of A) immobilized ubiquitin to E. coli ACP, B) immobilized ubiquitin to the ACP helix II peptide, and C) immobilized E. coli AcpS to Hrs UIM. The color coding of different sensorgram corresponds to the concentrations mentioned on the right side of the figure, used to pass over the immobilised sample. The plot of maximum response reached at equilibrium (Req) for each concentration of ligand used is shown in inset for each of the SPR sensorgram. Figure 9. Saturation transfer difference results for the ubiquitin- helix II peptide interaction. A) A region of the 1H TOCSY spectra of the helix II peptide (blue), overlaid on the 1H NOESY spectra (red). HN to H-1 NOE connectivities are shown, that were used to assign the backbone. B) 1H NMR Reference spectra for the helix II peptide (blue), stacked above the STD spectrum for ubiquitin complexed to helix II peptide in 1:8 molar ratio (colored black). The spectra were acquired at 298 K.


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B

A

1F80: ACP-AcpS

2D3G: Ubq-Hrs UIM

D

C

4ZJB: ACP-FabZ 4ETW: ACP-Pimelylmethyl ester esterase

E

F

4KEH: ACP-FabA ACS Paragon Plus Environment Figure 1

2FHS: ACP-Fab I

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Biochemistry

A Leu

Asp

Asp

Val

Glu

Val

Met

Glu

Glu Glu

B

E48 Ser

β1 I44

β2

Ala V43

Val M44

Leu V40

β4

β5 Glu

Glu S36

Hrs UIM: 258QEEEELALALSQSEAEEK277

Figure 2 ACS Paragon Plus Environment

D35

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A) HsHrs (258-277) HsSTAM1 (171-190) HsSTAM2 (165-184) HsUSP25 (97-116) HsUSP25 (124-140) HsATXN3 (224-243) HsATXN3 (244-263) HsATXN3 (335-354) HsAIRAPL(197-216) HsAIRAPL(221-240) HsRAP80 (80-99) HsRAP80 (105-124) ScUFO1 (546-566) ScUFO1 (583-602) ScUFO1 (651-668) ScVPS27 (257-277) ScVPS27 (301-320) HsUSP37 (704-723) HsUSP37 (806-825) HsUSP37 (828-847) MmRPN10 (211-230) MmRPN10 (282-301 HsParp10(650-670) HsParp10(673-693) HsAKIB1 (851-870) ScEPSIN1(165-184) ScEPSIN1(189-208) ScEPSIN2(175-194) ScEPSIN2(206-225) HsEPSIN1(183-202) HsEPSIN1(208-227) HsEPSIN2(275-294) HsEPSIN2(275-294) MmEPS15 (852-871) MmEPS15 (878-897) AtDAR1 (43-61) AtDAR1 (87-106) AtDAR1 (149-168) ScMET4 (139-157) MmDNMT1 (381-400) HsTRAC-1(210-224) HsRabex5(81-62) HsPIPSL (696-715) HsPIPSL (766-783) HsUCH-L3(216-230) HsGGA1 (271-288) HsTOM1 (275-292) HsGs (1-14) Consensus

-----QEEEELALALSQSEAEEK------- (O14964) -----KEEEDLAKAIELSLKEQRQQ----- (Q92783) -----KEDEDIAKAIELSLQEQKQQ----- (O75886) -----DDKDDLQRAIALSLAESNRA----- (Q9UHP3) -----DEEQAISRVLEASIAEN-------- (Q9UHP3) -----EDEEDLQRALALSRQEIDME----- (P54252) -----DEEADLRRAIQLSMQGSSRS----- (P54252) -----SEEDMLQAAVTMSLETVRND----- (P54252) -----SEDEALQRALEMSLAETKPQ----- (Q8WV99) -----QEEDDLALAQALSASE AEYQ-----(Q8WV99) -----TEEEQFALALKMSEQEAREV----- (Q96RL1) -----EEEELLRKAIAESLNSCRPS----- (Q96RL1) -----DEDEQLRRALEESQLIYETQ----- (Q04511) -----EDDEEFLRAIRQSRVEDERR----- (Q04511) -----NVDEDLQLAIALSLSEIN------- (Q04511) ----EDEEELIRKAIELSLKESRNS----- (P40343) -----EEDPDLKAAIQESLREAEEA----- (P40343) -----SEEELLAAVLEISKRDASPS------ (Q86T82) -----REEQELQQALAQSLQEQEAW----- (Q86T82) -----KEDDDLKRATELSLQEFNNS----- (Q86T82) -----SADPELALALRVSMEEQRQR----- (035226) -----TEEEQIAYAMQMSLQGTEFS----- (035226) ----LEEEAALQLALHRSLEPQGQY----- (Q53GL7) ----QEEAAALRQALTLSLLEQPPL----- (Q53GL7) -----EDDPNILLAIQLSLQESGLA----- (Q9P2G1) -----ENDDDLQRAISASRLTAEED----- (Q12518) -----KQDEDYETALQLSKEEEELK----- (Q12518) -----SYQDDLEKALEESRITAQED----- (Q05785) -----DEDPDFQAALQLSKEEEELK----- (Q05785) -----EEELQLQLALAMSKEEADQE----- (Q9Y6I3) -----EDDVQLQLALSLSREEHDKE----- (Q9Y6I3) -----EEELQLQLALAMSREVAEQE----- (O95208) -----GDDLRLQMALEESRRDTVKI----- (O95208) -----SEEDMIEWAKRESEREEEQR----- (P42567) -----QEQEDLELAIALSKSEISEA----- (P42567) ----DKEEIECAIALSLSEQEHV------- (Q8W4F0) -----DEDEEYMRAQLEAAEEEE------- (Q8W4F0) -----EEDELLAKALQESMNVGSP------ (Q8W4F0) -------DLDEQLAIELSAFADDSFI---- (P32389) -----VDEPQMLTSEKLSIYDSTST----- (P13864) ------EEALIRRVLDRSLLE--------- (Q96EQ8) ----EEEERQLREALEWDEQIQKQ------ (Q9UJ41-4) -----SADPELALVLRVFMEEQRQR----- (A2A3N6) ----MTEEEKIVCAMQMSLQGA-------- (A2A3N6) -----DPDELRFNAIALSAA-----------(P15374) ----EDNDEALAEILQANDNLT---------(Q9UJY5) ----IANEQLTEELLIVNDNLN---------(O60784) -----------MGCLGNSKTEDQRN------(p63092) -AcAcAcAcXXAXXSXXAc--------

------------------------------------------------------------------------------------------------------

B) E. coli V. harveyi Y. pestis S. synechocystis P. aeruginosa H. influenzae A. tumefaciens B. subtilis C. elegans P. falciparum H. pylori T. maritima L. major S. cerevisiae Consensus

35 DSLDTVELVMALEEE 49(P0A6A8) DSLDTVELVMALEEE (A0A0B4IT97) DSLDTVELVMALEEE (Q8ZFT4) DSLDTVELVMALEEE (P20804) DSLDTVELVMALEEE (O54439) DSLDVVELVMALEEE (P43709) DSLDTVELVMAFEEE (Q8UGE2) DSLDVVELVMELEDE (P80643) DSLDQVEIVMALEDE (D0BMS1) DSLDLVELIMALEEK (O77077) DSLDVVELIMALEEK (P56464) DSLDLVDLVMDFESE (Q9WZD0) NSLDVVEVVFAIEQE (E9AD06) DSLDTVELLVAIEEE (P32463) DSDXXXVXXEEE

Figure 3

ACS Paragon Plus Environment

Page 30 of 37

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D1

D3

D2

Loop I

0.4

15N

A

E30

D4

E. coli ACP + ubiquitin

D31 L32

B D38

0.3

'GAvgHN

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

E41

D31 E30

0.2

V43 L42

0.1

E41 V43 L46 D38 A45 E47

G12

I54

10

20

30

40

50

E57

A45 L46

E57 I62

E47

0.0 0

I62

G12

L32

60

70

80

Res. No.

Figure 4


I54

Biochemistry 1

1

2

A

2

3 4 15N

5 Ubiquitin + helix II peptide

V70 L73

F4

L69

D32 E34

Q49 L50

L43

R72 R74 G75

K27

(ppm)

T7 I13 T14

E18

15N

V5

AvgHN

15N

L71

G47

0.1

D

Ubiquitin +E. coli ACP K48

T12

D32

K29

T14

0.0

K48

B

Q49

Ubiquitin + helix II peptide

L71

K48

R72

D21

L69

L71

A28 D58

L73

0.1

AvgHN

I13

G47

Q49

V70

T14 I23 N25

T7 V5

L69

D32

L43 E34 Q41

E51

I61

1H

R72 L73 R74 G75

H68

(ppm)

I23 C1

E

I44 C1

0.0

I36 C1

C

Ubiquitin + Stam1 UIM I13 C1

(ppm)

K48 G47 F45

13C

0.4 vgHN

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Page 32 of 37

L8

I13

R42 L43

T7

0.2

R72 L73

T14

I44 C1 V70 C1

L71

V70 C2

Q49

13C

0.0 0

10

20

30 40 50 Res. No.

60

70

80

1H

K48 F45 I44 H68

L71

Ubiquitin + helix II peptide

K48 F45 I44 H68

L8

L8

L71

F

G

Ubq+Helix II peptide

Ubq+Stam1 UIM

Figure 5


(ppm)

Q62

Page 33 of 37

T66

Q40

F4

Q41

H68

L8

D32

V5

R74

T14

K48

I44 15N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Biochemistry

L71

Q49

L69

L43 L73 V70 L50 I13

Ubiquitin Free Ubiquitin + L. major ACP Ubiquitin + P. falciparum ACP Ubiquitin + Stam1 UIM peptide Ubiquitin + apo-EcACP Ubiquitin + holo-EcACP Ubiquitin + C8-EcACP

1H

Figure 6


Biochemistry

50

80

 Free ubiquitin  Ubiquitin+helix II peptide

A

Free ubiquitin

B

D52

40

60

30

20

L69

I3

T7 I13

K48

40

L69 R72 20

G10

10

R2eff [1/s]

G47

K48

E18

R72

0

0 0

10

20

30

40

50

60

0

70

100

200

300

400

500

600

700

v(CPMG) [Hz]

Res. No. 80

ubiquitin + ACP helix II peptide

C 70

R2eff [1/s]

Rex [1/s]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

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60

K48

50

L69

40

R72 30 0

100

200

300

400

v(CPMG) [Hz]

Figure 7


500

600

700

Page 35 of 37

60

40

Ubiquitin+ACP

30

50

B

40

Ubiquitin+helix II peptide

30

50

Req [RU]

A

Req [RU]

60

20

40

40 0 0

20

40

60

80

Concentration [M]

30

80 M 40 M 20 M 10 M 6.4 M 1 M

20

10

Response [RU]

10

20

10

0 0

30

100

200

300


20

315 M 270 M 225 M 180 M 90 M 45 M

10

0

-10

0 100

200

300

0

400

100

200

Time [sec]

Time [sec]

C

100

60

AcpS + Hrs UIM

50

Req [RU]

0

80

Response [RU]

Response [RU]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Biochemistry

40 30 20 10

60

0 0

100

200

300

400


40

300 M 250 M 200 M

20 0

100 M

-20 0

100

200

300

Time [sec]

Figure 8


400

300

400

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 36 of 37

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The 'recognition helix' of the type II acyl Carrier Protein (ACP) utilizes a 'ubiquitin interacting motif (UIM)' like surface to bind its partners

Ubiquitin

Met Val

Leu

Leu

Val

Ser

Ala

Glu

Glu

UIM

Ser

Glu

Helix II

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Biochemistry

Glu

ACP Helix II and UIM surface Ubiquitin-UIM/Helix II complex


of the Type II Acyl Carrier Protein (ACP) Utilizes a âUbiquitin

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