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NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP

NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP Miro2 and Miro1 are mitochondrial-associated proteins critical for regulating mitochondrial movement within the cell. Both Miro1 and Miro2 have roles in promoting neuron function, but recently Miro2 has been shown to have additional roles in response to nutrient starvation in tumor cells. Miro1 and 2 consist of two small GTPase domains a fl nking a pair of EF-hands. The N-terminal GTPase (nGTPase) domain is responsible for initiating mitochondrial trac ffi king and interactions with GCN1 in prostate cancer. The crystal structure of Miro1 nGTPase bound to GTP has been solved. However, no structural data is available for the nGTPase domain of Miro2. To better understand the similarities and differences in the functions of Miro1 and Miro2, we have initiated structural studies of Miro2. Here we report the backbone NMR chemical shift assignments of a 22 KDa construct of the nGTPase domain of Miro2 bound to GTP that includes residues 1–180 of the full-length protein. We affirm that the overall secondary structure of this complex closely resembles that of Miro1 nGTPase bound to GTP. Minor variations in the overall structures can be attributed to crystal packing interactions in the structure of Miro1. These NMR studies will form the foundation for future work identifying the specific interaction sites between Miro2 and its cel- lular binding partners. Keywords Miro2 N-terminal GTPase domain · Mitochondria · Solution state nuclear magnetic resonance · Backbone and sidechain nuclear magnetic resonance assignments · Chemical shifts Biological context a fl nking two EF-hand domains (Fig.  1) and are constitutively linked to mitochondria through a C-terminal transmem- Miro1 and Miro2 regulate the proper spatial distribution of brane domain. Mutation experiments show that the nGT- mitochondria in response to stimuli. The function of these Pase domain is critical for initiating mitochondrial traffick - proteins has been most studied in neurons but has also been ing in response to stimuli (Babic et al. 2015). Typically, the characterized in other cell types (Desai et al. 2013). Dys- release of GDP and subsequent binding of GTP induces a function of Miro1 in neurons has been linked to neurode- conformational change in small GTPase proteins leading to generation (Panchal and Tiwari 2021), while more recently, an increased affinity for an effector protein (Spoerner et al. Miro2 was found to have additional roles in the progression 2001), eliciting a downstream cellular response (Aspenstrom of prostate cancer (Furnish et al. 2022). 1999). Miro nGTPase domains have been classified as atypi- Miro1 and Miro2 are multidomain proteins contain- cal (Fransson et al. 2003), creating skepticism about their ing two small GTPase domains, (nGTPase and cGTPase), ability to function like canonical small GTPases. However, mutations that are proposed to mimic the GTP- and GDP- bound conformations display distinct cellular phenotypes * David N. M. Jones (Babic et al. 2015), suggesting that the nGTPase domains David.Jones@cuanschutz.edu have retained canonical function. The activity and interactions of small GTPases are regu- Program in Structural Biology and Biochemistry, University of Colorado School of Medicine, Anschutz Medical Campus, lated by five conserved motifs (named G1–G5) that allow the Aurora, CO 80045, USA protein to orchestrate conformational changes in response to Department of Pharmacology, University of Colorado School the binding of GDP or GTP and select for and tightly bind of Medicine, Anschutz Medical Campus, Aurora, CO 80045, to the guanine base. The function of each of these motifs USA Vol.:(0123456789) 1 3 350 C. E. Smith, D. N. M. Jones Fig. 1 Domain structure of Miro2 Miro2 contains two small GTPase domains at the N- and C termini flanking two EF-hand domains. A trans- membrane C-terminal helix anchors the protein to the outer mitochondrial membrane was first studied in H-Ras (Pai et al. 1989) and has been most likely a result of packing contacts formed in the crys- upheld by subsequent studies of other related canonical tals of Miro1. small GTPases that have retained all five motifs. In canonical small GTPases, residues in the rigid G1 loop motif (also referred to as the P loop) directly contact the Methods and experiments nucleotide phosphates and mitigate the effect of the addi- tional charges from the phosphates (Saraste et  al. 1990) Protein expression and purification Motifs G4 and G5 provide multiple direct interactions with the guanine base and the ribose to ensure that the nucleotide To obtain GTP-bound nGTPase, residues 1–180 of RhoT2 is bound tightly in the pocket (Pai et al. 1989) and to provide (Miro2) cDNA (Sinobiological) were PCR amplified and selectivity for guanine (Rensland et al. 1995; Vincent et al. subcloned into the Nde1/XhoI site of the pET28a expres- 2007). These regions do not show conformational changes sion vector, creating an N-terminal His -tag for purification. between GDP- and GTP-bound form. In contrast, residues in The plasmid was transformed into Escherichia coli BL21 the G2 motif (also referred to as switch I) and the G3 motif (DE3) competent cells for overexpression. Cultures were (switch II) differ significantly between the GTP- and GDP- grown in Luria broth at 37° C to O D = 0.6. Cells were bound conformations (Goody et al. 1992). The GDP-bound harvested at 3000 rpm and resuspended in labeled media for 13 15 form is often highly dynamic (Mello et al. 1997), and the expression. Double labeled ( C–  N) protein was expressed 15 13 binding of GTP induces a conformational change through in M9 media containing 1 g/L NH Cl and 2 g/L C-glu- 2 13 15 contacts with the gamma phosphate (Spoerner et al. 2001; cose, and H– C–  N labeled protein was expressed in M9 15 13 Hall et al. 2002), which creates a stable binding interface for media containing 1 g/L NH Cl and 2 g/L C-glucose in the effector protein (Milburn et al. 1990).99.9% D O. Cultures were grown at 37 °C to OD = 0.8. 2 600 Miro1 and Miro2 nGTPases have retained the G1, G4, Protein expression was induced by adding 0.125 mM iso- and G5 motifs. However, they do not contain canonical propyl 1-thio-beta-d -galactopyranoside (IPTG), and cells G2 and G3 motifs, which should induce a conformational were grown for 6 h at 18 °C. Cells were harvested by cen- change and mediate effector binding (Eberhardt et al. 2020). trifugation at 5000 rpm for 15 min at 4 °C and resuspended Therefore, the exact mechanism by which Miro2 nGTPase in 50  mM HEPES, 500  mM NaCl, 1  mM DTT, 1  mM receives upstream stimuli to produce the downstream MgCl 5% sucrose, 10 mM imidazole, 0.2 mM GTP, pH 2, response of mitochondrial trafficking initiation is unknown. 7.5 and lysed by sonication on ice. The lysate was clarified Many proteins have been determined to interact directly with by centrifugation at 15,000 rpm for 20 min. The soluble Miro nGTPases (Macaskill et al. 2009; Oeding et al. 2018) fraction was loaded onto Ni–NTA column (GE Pharma), and recently the interaction of Miro2 with GCN1 was shown equilibrated with 50 mM HEPES, 500 mM NaCl, 1 mM to be critical in driving prostate cancer progression. This DTT, 1 mM MgCl , 5% sucrose, 10 mM imidazole, pH 7.5. interaction also occurs through the nGTPase domain as clini- The Ni–NTA resin was washed using a step gradient by cally relevant mutations in this domain significantly impact increasing the imidazole concentration from 10 to 25 mM the level of interaction (Furnish et al. 2022). and then to 50  mM. The protein was eluted in the same Our goal is to understand how interactions of Miro2 with buffer containing 250 mM imidazole and diluted fivefold in its downstream effectors contribute to its function. We aim 25 mM HEPES 1 mM DTT 1 mM MgCl , pH 7.4. The pro- to identify the specific sites of interaction of different effec- tein was then loaded onto a HiTrap Q column (GE Pharma) tors with Miro and how the binding of different nucleotides and eluted using a linear gradient against 25 mM HEPES, impacts these interactions. For this, we obtained the back- 1 M NaCl, 1 mM DTT, 1 mM MgCl , pH 7.4. Protein was bone assignments of Miro2 nGTPase bound to GTP using concentrated and further purified by size exclusion chroma- 13 15 2 13 15 a combination of C/  N and H/ C/  N labeled proteins. tography (Superdex 75, GE Healthcare) with buffer con- We show that the secondary structure closely resembles taining 25 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM Miro1 nGTPase-GTP and that observed differences between MgCl , 5% sucrose, pH 7.5. Protein purity was assessed by the Miro1 crystal structure and Miro2 solution structure are SDS-PAGE to be greater than 95%. Protein concentration 1 3 NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP 351 was determined by UV absorbance at 280 nm using a Nan- Figure 2b depicts two regions in the spectrum with signif- oDrop (Thermofisher) and a molar extinction coefficient of icant peak overlap. Many peaks in this area belong to either −1 −1 20,050  M  cm for a 1:1 complex with GTP. residues from the His purification tag, the unstructured N-terminal residues (1–4), or the unstructured C-terminal NMR spectroscopy residues (172–180). Data obtained from multiple protonated and deuterated samples allowed for unambiguous assign- For NMR measurements, the protein was concentrated to ment of nearly all backbone chemical shifts belonging to the 1 15 0.3–0.4 mM, and 0.2 mM GTP, 5 mM DTT, and 10% D O Miro2 nGTPase domain. 176 of 180 non-proline H–  N (v/v) were added immediately prior to data acquisition. NMR correlation peaks (97.8%) were assigned. 177 of 180 C’ experiments were performed at 25 ˚C on a Bruker Avance (carbonyl) peaks (98.3%) were assigned. 176 out of 180 Cβ Neo 600 MHz equipped with a cryoprobe. Assignments of peaks (97.8%) were assigned, and 177 out of 180 Cα peaks 13 13 13 the protein main-chain atoms were made using sensitivity (98.3%) were assigned. Only 34.2% of Cγ, Cδ, and Cε 1 15 enhanced versions of 2D H/  N-HSQC (Kay et al. 1992), could be assigned from the CCONH-TOCSY dataset. The 3D HNCO (Grzesiek and Bax 1992b, Muhandiram and Kay assignment data has been deposited into BMRB with acces- 1994), 3D HNCACO (Clubb et al. 1992), 3D HNCACB, sion number 51500. CBCA(CO)NH (Grzesiek and Bax 1992a) and (H)CC(CO) NH (Grzesiek et al. 1993) of protonated samples and 3D Prediction of secondary structure HN(CO)CA, HN(COCA)CB and HNCACB (Grzesiek and Bax 1992b; Yamazaki et al. 1994) of per-deuterated samples. The secondary structure of Miro2 was predicted using the For per-deuterated samples, no additional procedures were 1 15 13 13 13 assigned H,  N, C’, Cβ, and Cα chemical shifts using used to back exchange labile protons as cross peak for all both TALOS-N (Shen and Bax 2013) and CheSPI (Nielsen 1 15 resonances were observed in the H–  N HSQC with com- and Mulder 2021). The results of these two programs are in parable intensities as the non-deuterated samples. generally good agreement. The advantage of CheSPI is that All 3D experiments were collected using non-uniform it can provide insight into the relative structure and dynam- sampling methods (Barna et al. 1987) using the Poisson-gap ics and can discern the relative contributions of different sampling schemes implemented by Hyberts et al. (Hyberts types of structures that contribute to the chemical shifts. et al. 2010) and with a sampling density of 35–40%. Data Further, CheSPI can discriminate up to eight types of sec- were processed using NMRpipe (Delaglio et al. 1995) and ondary structure elements for structured proteins. We com- NUS data were reconstructed using SMILE (Hyberts et al. pared the predicted structure to that of the nGTPase of Miro1 2012, 2014), and resonance assignments were determined (Smith et al. 2020) (PDB 6d71). Figure 3a shows the amino using Ccpnmr Analysis v 2.4.2 (Vranken et al. 2005). acid sequence alignment of the nGTPase of human Miro1 and human Miro2 which have 72.78% identity. Figure 3b Extent of assignments and data depositions shows that the structure predictions using TALOS-N and CheSPI closely mirror that seen in the structure of Miro1 1 15 15 13 The assigned H–  N HSQC spectrum of  N/ C labled nGTPase bound to GTP (represented in the bar above chart). Miro2-nGTPase (residues 1–180) is shown in Fig. 2a. Resi- The CheSPI result in Fig. 3b shows the relative contributions dues belonging to the purification tag are labeled in paren- from the four major types of structure: helix (red), extended thesis, otherwise numbering of residues corresponds to the (blue), turn (green) and unstructured (grey). A significant Miro2 sequence. The spectrum exhibits good peak disper- deviation in the prediction for Miro2 compared to Miro1 is sion, indicating that it is well-folded in solution. The peak seen for residues 122 to 124, which adopt a helical structure corresponding to Gly11, located in the G1 motif involved in in Miro1 but are predicted to adopt a more extended con- nucleotide binding, exhibits an extreme H chemical shift at formation in Miro2. Inspection of the crystal structure of 4.54 ppm. In the crystal structure of Miro1 nGTPase, which Miro1 reveals that this region forms crystal contacts with shares a 73% sequence identity with Miro2, the amide proton a symmetry-related molecule, so the observed differences of Gly11 packs against the aromatic ring of Trp97 (d 2.9 Å), could arise from packing artifacts. However, confirmation of and this likely accounts for the extreme upfield shift of this these differences will require solution analysis of the Miro1 residue. These types of interactions have been identified for nGTPase domain. Having the GTP sample stable in solu- a large number of proteins as contributing to overall stability tion and the backbone assigned allows for future studies in (Toth et al. 2001). Additionally, Ala149 has a  N chemical characterizing the interaction of this important protein with shift of 135.04. This residue is in the G5 motif and canoni- its binding partners. cally hydrogen bonds with the O6 atom of the guanine ring (Pai et al. 1989), which is also found in the Miro1 structure. 1 3 352 C. E. Smith, D. N. M. Jones Fig. 2 NMR assignments of Miro2 nGTPase a Two- 1 15 dimensional H–  N HSQC 15 13 spectrum of  N/ C labeled protein showing the residue assignments of Miro2 nGTPase- GTP complex, The inlaid region shows the peak from Gly11, which has an extreme H chemi- cal shift at ~ 4.55 ppm. Residues belonging to the purification tag are labeled in parentheses. b Enlargements of the overlapped regions indicated with boxes in panel a showing the assign- ments in greater detail 1 3 NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP 353 Fig. 3 Comparison of Miro2 with Miro1 a Amino acid sequence tive contributions for the four major types of structure to the chemi- alignment of hMiro1 and hMiro2. The secondary structure ele- cal shifts are presented as a stacked plot with helix indicated in red, ments observed in the crystal structure of hMiro1-GTP are indicated extended(blue), turn (green), and non-structured (grey) The bar above above. b Chemical shift-based secondary structure prediction of the chart depicts the known secondary structure elements in the the Miro2 nGTPase bound to GTP using TALOS-N (upper panel) deposited structure of Miro1 nGTPase bound to GTP (PDB: 6d71) and CheSPI (lower panel). For TALOS probabilities for helices are Helix in red and extended structure indicated in blue) indicated in red and extended structure in blue. For CheSPI the rela- 1 3 354 C. E. Smith, D. N. M. Jones Acknowledgements The operation of the NMR facilities at CU School Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) of Medicine is supported by the University of Colorado Cancer Center NMRPipe: a multidimensional spectral processing system based Support Grant National Institute of Health 5P30-CA046934 and on UNIX pipes. J Biomol NMR 6:277–293. https:// doi. org/ 10. National Institute of Health grant S10 OD025020.1007/ bf001 97809 Desai SP, Bhatia SN, Toner M, Irimia D (2013) Mitochondrial localiza- tion and the persistent migration of epithelial cancer cells. Bio- Author contributions CS and DJ designed the experiments; CS pre- phys J 104:2077–2088. https:// doi. org/ 10. 1016/j. bpj. 2013. 03. 02 pared the samples; CS and DJ collected the NMR experiments; CS Eberhardt EL, Ludlam AV, Tan Z, Cianfrocco MA (2020) Miro: a analyzed the experiments; CS and DJ discussed the results, wrote and molecular switch at the center of mitochondrial regulation. Protein edited the manuscript and approved the final version. Sci 29:1269–1284. https:// doi. org/ 10. 1002/ pro. 3839 Fransson A, Ruusala A, Aspenstrom P (2003) Atypical Rho GTPases Funding The operation of the NMR facilities at CU School of Medi- have roles in mitochondrial homeostasis and apoptosis. J Biol cine is supported by the University of Colorado Cancer Center Sup- Chem 278:6495–6502. https:// doi. org/ 10. 1074/ jbc. M2086 09200 port Grant National Institute of Health 5P30-CA046934 and National Furnish M, Boulton DP, Genther V, Grofova D, Ellinwood ML, Romero Institute of Health Grant S10 OD025020. L, Lucia MS, Cramer SD, Caino MC (2022) MIRO2 regulates prostate cancer cell growth via GCN1-dependent stress signaling. Data availability The chemical shift assignments of Miro2 nGTPase- Mol Cancer Res 20:607–621. https://doi. or g/10. 1158/ 1541- 7786. GTP have been deposited into BMRB with Accession Number 51500. MCR- 21- 0374 Goody RS, Pai EF, Schlichting I, Rensland H, Scheidig A, Franken S, Wittinghofer A (1992) Studies on the structure and mechanism Declarations of H-Ras p21. 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Trends Biochem Sci Publisher's Note Springer Nature remains neutral with regard to 15:430–434. https:// doi. org/ 10. 1016/ 0968- 0004(90) 90281-f jurisdictional claims in published maps and institutional affiliations. Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biomolecular NMR Assignments Springer Journals

NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP

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Abstract

Miro2 and Miro1 are mitochondrial-associated proteins critical for regulating mitochondrial movement within the cell. Both Miro1 and Miro2 have roles in promoting neuron function, but recently Miro2 has been shown to have additional roles in response to nutrient starvation in tumor cells. Miro1 and 2 consist of two small GTPase domains a fl nking a pair of EF-hands. The N-terminal GTPase (nGTPase) domain is responsible for initiating mitochondrial trac ffi king and interactions with GCN1 in prostate cancer. The crystal structure of Miro1 nGTPase bound to GTP has been solved. However, no structural data is available for the nGTPase domain of Miro2. To better understand the similarities and differences in the functions of Miro1 and Miro2, we have initiated structural studies of Miro2. Here we report the backbone NMR chemical shift assignments of a 22 KDa construct of the nGTPase domain of Miro2 bound to GTP that includes residues 1–180 of the full-length protein. We affirm that the overall secondary structure of this complex closely resembles that of Miro1 nGTPase bound to GTP. Minor variations in the overall structures can be attributed to crystal packing interactions in the structure of Miro1. These NMR studies will form the foundation for future work identifying the specific interaction sites between Miro2 and its cel- lular binding partners. Keywords Miro2 N-terminal GTPase domain · Mitochondria · Solution state nuclear magnetic resonance · Backbone and sidechain nuclear magnetic resonance assignments · Chemical shifts Biological context a fl nking two EF-hand domains (Fig.  1) and are constitutively linked to mitochondria through a C-terminal transmem- Miro1 and Miro2 regulate the proper spatial distribution of brane domain. Mutation experiments show that the nGT- mitochondria in response to stimuli. The function of these Pase domain is critical for initiating mitochondrial traffick - proteins has been most studied in neurons but has also been ing in response to stimuli (Babic et al. 2015). Typically, the characterized in other cell types (Desai et al. 2013). Dys- release of GDP and subsequent binding of GTP induces a function of Miro1 in neurons has been linked to neurode- conformational change in small GTPase proteins leading to generation (Panchal and Tiwari 2021), while more recently, an increased affinity for an effector protein (Spoerner et al. Miro2 was found to have additional roles in the progression 2001), eliciting a downstream cellular response (Aspenstrom of prostate cancer (Furnish et al. 2022). 1999). Miro nGTPase domains have been classified as atypi- Miro1 and Miro2 are multidomain proteins contain- cal (Fransson et al. 2003), creating skepticism about their ing two small GTPase domains, (nGTPase and cGTPase), ability to function like canonical small GTPases. However, mutations that are proposed to mimic the GTP- and GDP- bound conformations display distinct cellular phenotypes * David N. M. Jones (Babic et al. 2015), suggesting that the nGTPase domains David.Jones@cuanschutz.edu have retained canonical function. The activity and interactions of small GTPases are regu- Program in Structural Biology and Biochemistry, University of Colorado School of Medicine, Anschutz Medical Campus, lated by five conserved motifs (named G1–G5) that allow the Aurora, CO 80045, USA protein to orchestrate conformational changes in response to Department of Pharmacology, University of Colorado School the binding of GDP or GTP and select for and tightly bind of Medicine, Anschutz Medical Campus, Aurora, CO 80045, to the guanine base. The function of each of these motifs USA Vol.:(0123456789) 1 3 350 C. E. Smith, D. N. M. Jones Fig. 1 Domain structure of Miro2 Miro2 contains two small GTPase domains at the N- and C termini flanking two EF-hand domains. A trans- membrane C-terminal helix anchors the protein to the outer mitochondrial membrane was first studied in H-Ras (Pai et al. 1989) and has been most likely a result of packing contacts formed in the crys- upheld by subsequent studies of other related canonical tals of Miro1. small GTPases that have retained all five motifs. In canonical small GTPases, residues in the rigid G1 loop motif (also referred to as the P loop) directly contact the Methods and experiments nucleotide phosphates and mitigate the effect of the addi- tional charges from the phosphates (Saraste et  al. 1990) Protein expression and purification Motifs G4 and G5 provide multiple direct interactions with the guanine base and the ribose to ensure that the nucleotide To obtain GTP-bound nGTPase, residues 1–180 of RhoT2 is bound tightly in the pocket (Pai et al. 1989) and to provide (Miro2) cDNA (Sinobiological) were PCR amplified and selectivity for guanine (Rensland et al. 1995; Vincent et al. subcloned into the Nde1/XhoI site of the pET28a expres- 2007). These regions do not show conformational changes sion vector, creating an N-terminal His -tag for purification. between GDP- and GTP-bound form. In contrast, residues in The plasmid was transformed into Escherichia coli BL21 the G2 motif (also referred to as switch I) and the G3 motif (DE3) competent cells for overexpression. Cultures were (switch II) differ significantly between the GTP- and GDP- grown in Luria broth at 37° C to O D = 0.6. Cells were bound conformations (Goody et al. 1992). The GDP-bound harvested at 3000 rpm and resuspended in labeled media for 13 15 form is often highly dynamic (Mello et al. 1997), and the expression. Double labeled ( C–  N) protein was expressed 15 13 binding of GTP induces a conformational change through in M9 media containing 1 g/L NH Cl and 2 g/L C-glu- 2 13 15 contacts with the gamma phosphate (Spoerner et al. 2001; cose, and H– C–  N labeled protein was expressed in M9 15 13 Hall et al. 2002), which creates a stable binding interface for media containing 1 g/L NH Cl and 2 g/L C-glucose in the effector protein (Milburn et al. 1990).99.9% D O. Cultures were grown at 37 °C to OD = 0.8. 2 600 Miro1 and Miro2 nGTPases have retained the G1, G4, Protein expression was induced by adding 0.125 mM iso- and G5 motifs. However, they do not contain canonical propyl 1-thio-beta-d -galactopyranoside (IPTG), and cells G2 and G3 motifs, which should induce a conformational were grown for 6 h at 18 °C. Cells were harvested by cen- change and mediate effector binding (Eberhardt et al. 2020). trifugation at 5000 rpm for 15 min at 4 °C and resuspended Therefore, the exact mechanism by which Miro2 nGTPase in 50  mM HEPES, 500  mM NaCl, 1  mM DTT, 1  mM receives upstream stimuli to produce the downstream MgCl 5% sucrose, 10 mM imidazole, 0.2 mM GTP, pH 2, response of mitochondrial trafficking initiation is unknown. 7.5 and lysed by sonication on ice. The lysate was clarified Many proteins have been determined to interact directly with by centrifugation at 15,000 rpm for 20 min. The soluble Miro nGTPases (Macaskill et al. 2009; Oeding et al. 2018) fraction was loaded onto Ni–NTA column (GE Pharma), and recently the interaction of Miro2 with GCN1 was shown equilibrated with 50 mM HEPES, 500 mM NaCl, 1 mM to be critical in driving prostate cancer progression. This DTT, 1 mM MgCl , 5% sucrose, 10 mM imidazole, pH 7.5. interaction also occurs through the nGTPase domain as clini- The Ni–NTA resin was washed using a step gradient by cally relevant mutations in this domain significantly impact increasing the imidazole concentration from 10 to 25 mM the level of interaction (Furnish et al. 2022). and then to 50  mM. The protein was eluted in the same Our goal is to understand how interactions of Miro2 with buffer containing 250 mM imidazole and diluted fivefold in its downstream effectors contribute to its function. We aim 25 mM HEPES 1 mM DTT 1 mM MgCl , pH 7.4. The pro- to identify the specific sites of interaction of different effec- tein was then loaded onto a HiTrap Q column (GE Pharma) tors with Miro and how the binding of different nucleotides and eluted using a linear gradient against 25 mM HEPES, impacts these interactions. For this, we obtained the back- 1 M NaCl, 1 mM DTT, 1 mM MgCl , pH 7.4. Protein was bone assignments of Miro2 nGTPase bound to GTP using concentrated and further purified by size exclusion chroma- 13 15 2 13 15 a combination of C/  N and H/ C/  N labeled proteins. tography (Superdex 75, GE Healthcare) with buffer con- We show that the secondary structure closely resembles taining 25 mM HEPES, 150 mM NaCl, 1 mM DTT, 1 mM Miro1 nGTPase-GTP and that observed differences between MgCl , 5% sucrose, pH 7.5. Protein purity was assessed by the Miro1 crystal structure and Miro2 solution structure are SDS-PAGE to be greater than 95%. Protein concentration 1 3 NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP 351 was determined by UV absorbance at 280 nm using a Nan- Figure 2b depicts two regions in the spectrum with signif- oDrop (Thermofisher) and a molar extinction coefficient of icant peak overlap. Many peaks in this area belong to either −1 −1 20,050  M  cm for a 1:1 complex with GTP. residues from the His purification tag, the unstructured N-terminal residues (1–4), or the unstructured C-terminal NMR spectroscopy residues (172–180). Data obtained from multiple protonated and deuterated samples allowed for unambiguous assign- For NMR measurements, the protein was concentrated to ment of nearly all backbone chemical shifts belonging to the 1 15 0.3–0.4 mM, and 0.2 mM GTP, 5 mM DTT, and 10% D O Miro2 nGTPase domain. 176 of 180 non-proline H–  N (v/v) were added immediately prior to data acquisition. NMR correlation peaks (97.8%) were assigned. 177 of 180 C’ experiments were performed at 25 ˚C on a Bruker Avance (carbonyl) peaks (98.3%) were assigned. 176 out of 180 Cβ Neo 600 MHz equipped with a cryoprobe. Assignments of peaks (97.8%) were assigned, and 177 out of 180 Cα peaks 13 13 13 the protein main-chain atoms were made using sensitivity (98.3%) were assigned. Only 34.2% of Cγ, Cδ, and Cε 1 15 enhanced versions of 2D H/  N-HSQC (Kay et al. 1992), could be assigned from the CCONH-TOCSY dataset. The 3D HNCO (Grzesiek and Bax 1992b, Muhandiram and Kay assignment data has been deposited into BMRB with acces- 1994), 3D HNCACO (Clubb et al. 1992), 3D HNCACB, sion number 51500. CBCA(CO)NH (Grzesiek and Bax 1992a) and (H)CC(CO) NH (Grzesiek et al. 1993) of protonated samples and 3D Prediction of secondary structure HN(CO)CA, HN(COCA)CB and HNCACB (Grzesiek and Bax 1992b; Yamazaki et al. 1994) of per-deuterated samples. The secondary structure of Miro2 was predicted using the For per-deuterated samples, no additional procedures were 1 15 13 13 13 assigned H,  N, C’, Cβ, and Cα chemical shifts using used to back exchange labile protons as cross peak for all both TALOS-N (Shen and Bax 2013) and CheSPI (Nielsen 1 15 resonances were observed in the H–  N HSQC with com- and Mulder 2021). The results of these two programs are in parable intensities as the non-deuterated samples. generally good agreement. The advantage of CheSPI is that All 3D experiments were collected using non-uniform it can provide insight into the relative structure and dynam- sampling methods (Barna et al. 1987) using the Poisson-gap ics and can discern the relative contributions of different sampling schemes implemented by Hyberts et al. (Hyberts types of structures that contribute to the chemical shifts. et al. 2010) and with a sampling density of 35–40%. Data Further, CheSPI can discriminate up to eight types of sec- were processed using NMRpipe (Delaglio et al. 1995) and ondary structure elements for structured proteins. We com- NUS data were reconstructed using SMILE (Hyberts et al. pared the predicted structure to that of the nGTPase of Miro1 2012, 2014), and resonance assignments were determined (Smith et al. 2020) (PDB 6d71). Figure 3a shows the amino using Ccpnmr Analysis v 2.4.2 (Vranken et al. 2005). acid sequence alignment of the nGTPase of human Miro1 and human Miro2 which have 72.78% identity. Figure 3b Extent of assignments and data depositions shows that the structure predictions using TALOS-N and CheSPI closely mirror that seen in the structure of Miro1 1 15 15 13 The assigned H–  N HSQC spectrum of  N/ C labled nGTPase bound to GTP (represented in the bar above chart). Miro2-nGTPase (residues 1–180) is shown in Fig. 2a. Resi- The CheSPI result in Fig. 3b shows the relative contributions dues belonging to the purification tag are labeled in paren- from the four major types of structure: helix (red), extended thesis, otherwise numbering of residues corresponds to the (blue), turn (green) and unstructured (grey). A significant Miro2 sequence. The spectrum exhibits good peak disper- deviation in the prediction for Miro2 compared to Miro1 is sion, indicating that it is well-folded in solution. The peak seen for residues 122 to 124, which adopt a helical structure corresponding to Gly11, located in the G1 motif involved in in Miro1 but are predicted to adopt a more extended con- nucleotide binding, exhibits an extreme H chemical shift at formation in Miro2. Inspection of the crystal structure of 4.54 ppm. In the crystal structure of Miro1 nGTPase, which Miro1 reveals that this region forms crystal contacts with shares a 73% sequence identity with Miro2, the amide proton a symmetry-related molecule, so the observed differences of Gly11 packs against the aromatic ring of Trp97 (d 2.9 Å), could arise from packing artifacts. However, confirmation of and this likely accounts for the extreme upfield shift of this these differences will require solution analysis of the Miro1 residue. These types of interactions have been identified for nGTPase domain. Having the GTP sample stable in solu- a large number of proteins as contributing to overall stability tion and the backbone assigned allows for future studies in (Toth et al. 2001). Additionally, Ala149 has a  N chemical characterizing the interaction of this important protein with shift of 135.04. This residue is in the G5 motif and canoni- its binding partners. cally hydrogen bonds with the O6 atom of the guanine ring (Pai et al. 1989), which is also found in the Miro1 structure. 1 3 352 C. E. Smith, D. N. M. Jones Fig. 2 NMR assignments of Miro2 nGTPase a Two- 1 15 dimensional H–  N HSQC 15 13 spectrum of  N/ C labeled protein showing the residue assignments of Miro2 nGTPase- GTP complex, The inlaid region shows the peak from Gly11, which has an extreme H chemi- cal shift at ~ 4.55 ppm. Residues belonging to the purification tag are labeled in parentheses. b Enlargements of the overlapped regions indicated with boxes in panel a showing the assign- ments in greater detail 1 3 NMR resonance assignment of the N-terminal GTPase domain of human Miro2 Bound to GTP 353 Fig. 3 Comparison of Miro2 with Miro1 a Amino acid sequence tive contributions for the four major types of structure to the chemi- alignment of hMiro1 and hMiro2. The secondary structure ele- cal shifts are presented as a stacked plot with helix indicated in red, ments observed in the crystal structure of hMiro1-GTP are indicated extended(blue), turn (green), and non-structured (grey) The bar above above. b Chemical shift-based secondary structure prediction of the chart depicts the known secondary structure elements in the the Miro2 nGTPase bound to GTP using TALOS-N (upper panel) deposited structure of Miro1 nGTPase bound to GTP (PDB: 6d71) and CheSPI (lower panel). For TALOS probabilities for helices are Helix in red and extended structure indicated in blue) indicated in red and extended structure in blue. For CheSPI the rela- 1 3 354 C. E. Smith, D. N. M. Jones Acknowledgements The operation of the NMR facilities at CU School Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) of Medicine is supported by the University of Colorado Cancer Center NMRPipe: a multidimensional spectral processing system based Support Grant National Institute of Health 5P30-CA046934 and on UNIX pipes. J Biomol NMR 6:277–293. https:// doi. org/ 10. National Institute of Health grant S10 OD025020.1007/ bf001 97809 Desai SP, Bhatia SN, Toner M, Irimia D (2013) Mitochondrial localiza- tion and the persistent migration of epithelial cancer cells. 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Biomolecular NMR AssignmentsSpringer Journals

Published: Oct 1, 2022

Keywords: Miro2 N-terminal GTPase domain; Mitochondria; Solution state nuclear magnetic resonance; Backbone and sidechain nuclear magnetic resonance assignments; Chemical shifts

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