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Flores, Samuel
Alternative names
Publications (10 of 24) Show all publications
Nosrati, M., Solbak, S., Nordesjö, O., Nissbeck, M., Dourado, D. F. A., Andersson, K. G., . . . Flores, S. C. (2017). Insights from engineering the Affibody-Fc interaction with a computational-experimental method. Protein Engineering Design & Selection, 30(9), 593-601
Open this publication in new window or tab >>Insights from engineering the Affibody-Fc interaction with a computational-experimental method
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2017 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 30, no 9, p. 593-601Article in journal (Refereed) Published
Abstract [en]

The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.

Keywords
Staphylococcal Protein A, affinity, computational prediction, protein-protein interactions, surface plasmon resonance
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-337576 (URN)10.1093/protein/gzx023 (DOI)000413767000004 ()28472513 (PubMedID)
Funder
Swedish Research Council, D0571301
Available from: 2018-01-02 Created: 2018-01-02 Last updated: 2018-02-16Bibliographically approved
Tek, A., Korostelev, A. A. & Flores, S. C. (2016). MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory. Nucleic Acids Research, 44(1), 95-105
Open this publication in new window or tab >>MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory
2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 1, p. 95-105Article in journal (Refereed) Published
Abstract [en]

Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-282497 (URN)10.1093/nar/gkv1457 (DOI)000371264000016 ()26673695 (PubMedID)
Funder
NIH (National Institute of Health), R01 GM106105eSSENCE - An eScience Collaboration
Available from: 2016-04-05 Created: 2016-04-05 Last updated: 2017-11-30Bibliographically approved
Dourado, D. F. A. & Flores, S. C. (2016). Modeling and fitting protein-protein complexes to predict change of binding energy. Scientific Reports, 6, Article ID 25406.
Open this publication in new window or tab >>Modeling and fitting protein-protein complexes to predict change of binding energy
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 25406Article in journal (Refereed) Published
Abstract [en]

It is possible to accurately and economically predict change in protein-protein interaction energy upon mutation (Delta Delta G), when a high-resolution structure of the complex is available. This is of growing usefulness for design of high-affinity or otherwise modified binding proteins for therapeutic, diagnostic, industrial, and basic science applications. Recently the field has begun to pursue Delta Delta G prediction for homology modeled complexes, but so far this has worked mostly for cases of high sequence identity. If the interacting proteins have been crystallized in free (uncomplexed) form, in a majority of cases it is possible to find a structurally similar complex which can be used as the basis for template-based modeling. We describe how to use MMB to create such models, and then use them to predict Delta Delta G, using a dataset consisting of free target structures, co-crystallized template complexes with sequence identify with respect to the targets as low as 44%, and experimental Delta Delta G measurements. We obtain similar results by fitting to a low-resolution Cryo-EM density map. Results suggest that other structural constraints may lead to a similar outcome, making the method even more broadly applicable.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-297785 (URN)10.1038/srep25406 (DOI)000375797500001 ()27173910 (PubMedID)
Funder
eSSENCE - An eScience CollaborationThe Swedish Foundation for International Cooperation in Research and Higher Education (STINT)
Available from: 2016-06-28 Created: 2016-06-28 Last updated: 2018-01-10Bibliographically approved
Koripella, R. K., Holm, M., Dourado, D., Mandava, C. S., Flores, S. & Sanyal, S. (2015). A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate. Scientific Reports, 5, Article ID 12970.
Open this publication in new window or tab >>A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12970Article in journal (Refereed) Published
Abstract [en]

Elongation factor G (EF-G), a translational GTPase responsible for tRNA-mRNA translocation possesses a conserved histidine (H91 in Escherichia coli) at the apex of switch-II, which has been implicated in GTPase activation and GTP hydrolysis. While H91A, H91R and H91E mutants showed different degrees of defect in ribosome associated GTP hydrolysis, H91Q behaved like the WT. However, all these mutants, including H91Q, are much more defective in inorganic phosphate (Pi) release, thereby suggesting that H91 facilitates Pi release. In crystal structures of the ribosome bound EF-G center dot GTP a tight coupling between H91 and the gamma-phosphate of GTP can be seen. Following GTP hydrolysis, H91 flips similar to 140 degrees in the opposite direction, probably with Pi still coupled to it. This, we suggest, promotes Pi to detach from GDP and reach the inter-domain space of EF-G, which constitutes an exit path for the Pi. Molecular dynamics simulations are consistent with this hypothesis and demonstrate a vital role of an Mg2+ ion in the process.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-261233 (URN)10.1038/srep12970 (DOI)000359373400001 ()
Funder
Swedish Research Council, 2011-6088 2014-4423 2008-6593Knut and Alice Wallenberg Foundation, KAW 2011.0081
Available from: 2015-09-07 Created: 2015-08-31 Last updated: 2017-12-04Bibliographically approved
Fiesel, F. C., Caulfield, T. R., Moussaud-Lamodiere, E. L., Ogaki, K., Dourado, D. F. A., Flores, S. C., . . . Springer, W. (2015). Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin. Human Mutation, 36(8), 774-786
Open this publication in new window or tab >>Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin
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2015 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 36, no 8, p. 774-786Article in journal (Refereed) Published
Abstract [en]

Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.

Keywords
PARK2, PINK1, Parkinson, EOPD, mitophagy, molecular dynamics
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-260607 (URN)10.1002/humu.22808 (DOI)000358376600007 ()25939424 (PubMedID)
Funder
NIH (National Institute of Health), NS085070, NS072187eSSENCE - An eScience Collaboration
Note

Contract grant sponsors: NIH/NINDS (R01 #NS085070); the Michael J. Fox Foundation for Parkinson's Research and the Foundation for Mitochondrial Medicine; Mayo Clinic Foundation and the Center for Individualized Medicine; the Marriott Family Foundation; Gerstner Family Career Development Award; NIH/NINDS (P50 #NS072187); Uppsala University and eSSENCE (essenceofscience.se); Wenner-Gren Foundation.

Available from: 2015-08-24 Created: 2015-08-21 Last updated: 2018-01-11Bibliographically approved
Dourado, D. F. A. & Flores, S. C. (2014). A multiscale approach to predicting affinity changes in protein-protein interfaces. Proteins: Structure, Function, and Bioinformatics, 82(10), 2681-2690
Open this publication in new window or tab >>A multiscale approach to predicting affinity changes in protein-protein interfaces
2014 (English)In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 82, no 10, p. 2681-2690Article in journal (Refereed) Published
Abstract [en]

Substitution mutations in protein-protein interfaces can have a substantial effect on binding, which has consequences in basic and applied biomedical research. Experimental expression, purification, and affinity determination of protein complexes is an expensive and time-consuming means of evaluating the effect of mutations, making a fast and accurate in silico method highly desirable. When the structure of the wild-type complex is known, it is possible to economically evaluate the effect of point mutations with knowledge based potentials, which do not model backbone flexibility, but these have been validated only for single mutants. Substitution mutations tend to induce local conformational rearrangements only. Accordingly, ZEMu (Zone Equilibration of Mutants) flexibilizes only a small region around the site of mutation, then computes its dynamics under a physics-based force field. We validate with 1254 experimental mutants (with 1-15 simultaneous substitutions) in a wide variety of different protein environments (65 protein complexes), and obtain a significant improvement in the accuracy of predicted Delta Delta G.

Keywords
biologic design, internal coordinate mechanics, multiscale modeling, affinity maturation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-236070 (URN)10.1002/prot.24634 (DOI)000342849400033 ()
Available from: 2014-11-12 Created: 2014-11-12 Last updated: 2018-01-26Bibliographically approved
Flores, S. C. (2014). Elucidating Ribosomal Translocation with Internal Coordinate Flexible Fitting. Paper presented at 58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA. Biophysical Journal, 106(2), 492A-493A
Open this publication in new window or tab >>Elucidating Ribosomal Translocation with Internal Coordinate Flexible Fitting
2014 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 492A-493AArticle in journal, Meeting abstract (Other academic) Published
Abstract [en]

Determining conformational changes of large macromolecules is challenging experimentally and computationally. The ribosome has been observed crystallographically in several states but many others have been seen only by low-resolution methods including cryo-electron microscopy. Meanwhile the crucial dynamics between states remain out of reach of experimental structure determination methods. Most existing computational approaches model complexes at all-atom resolution, at very high cost, or use approximations which lose some of the most interesting dynamical details. I have developed Internal Coordinate Flexible Fitting (ICFF), a multiscale method that uses full atomic forces and flexibility only in key regions of a model, capturing extensive conformational rearrangements at low cost. I use ICFF to turn low-resolution density maps, crystallographic structures, and biochemical information into the largest-scale all-atoms trajectory of ribosomal translocation modeled to date. ICFF is three orders of magnitude faster than the most comparable existing method. The results suggest an intriguing possible mechanism of translocation.

National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-228604 (URN)10.1016/j.bpj.2013.11.2754 (DOI)000337000402727 ()
Conference
58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA
Available from: 2014-07-18 Created: 2014-07-17 Last updated: 2017-12-05Bibliographically approved
Flores, S. C. (2014). Fast fitting to low resolution density maps: elucidating large-scale motions of the ribosome. Nucleic Acids Research, 42(2), e9
Open this publication in new window or tab >>Fast fitting to low resolution density maps: elucidating large-scale motions of the ribosome
2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 2, p. e9-Article in journal (Refereed) Published
Abstract [en]

Determining the conformational rearrangements of large macromolecules is challenging experimentally and computationally. Case in point is the ribosome; it has been observed by high-resolution crystallography in several states, but many others are known only from low-resolution methods including cryoelectron microscopy. Combining these data into dynamical trajectories that may aid understanding of its largest-scale conformational changes has so far remained out of reach of computational methods. Most existing methods either model all atoms explicitly, resulting in often prohibitive cost, or use approximations that lose interesting structural and dynamical detail. In this work, I introduce Internal Coordinate Flexible Fitting, which uses full atomic forces and flexibility in limited regions of a model, capturing extensive conformational rearrangements at low cost. I use it to turn multiple low-resolution density maps, crystallographic structures and biochemical information into unified all-atoms trajectories of ribosomal translocation. Internal Coordinate Flexible Fitting is three orders of magnitude faster than the most comparable existing method.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-221062 (URN)10.1093/nar/gkt906 (DOI)000331138100002 ()
Available from: 2014-03-26 Created: 2014-03-25 Last updated: 2017-12-05Bibliographically approved
Tek, A., Chen, Y., Selmer, M. & Flores, S. C. (2014). Investigating Ribosome Conformations with Multi-Resolution Modeling. Paper presented at 58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA. Biophysical Journal, 106(2), 491A-491A
Open this publication in new window or tab >>Investigating Ribosome Conformations with Multi-Resolution Modeling
2014 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 491A-491AArticle in journal, Meeting abstract (Other academic) Published
National Category
Biophysics
Identifiers
urn:nbn:se:uu:diva-228603 (URN)10.1016/j.bpj.2013.11.2744 (DOI)000337000402717 ()
Conference
58th Annual Meeting of the Biophysical-Society, FEB 15-19, 2014, San Francisco, CA
Available from: 2014-07-18 Created: 2014-07-17 Last updated: 2017-12-05Bibliographically approved
Caulfield, T. R., Fiesel, F. C., Moussaud-Lamodiere, E. L., Dourado, D. F. A., Flores, S. C. & Springer, W. (2014). Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin. PloS Computational Biology, 10(11), e1003935
Open this publication in new window or tab >>Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin
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2014 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 10, no 11, p. e1003935-Article in journal (Refereed) Published
Abstract [en]

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimalbiasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-240135 (URN)10.1371/journal.pcbi.1003935 (DOI)000345454400024 ()25375667 (PubMedID)
Available from: 2015-01-07 Created: 2015-01-05 Last updated: 2017-12-05Bibliographically approved
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