Publications by authors named "Cristina Puchades"

15 Publications

  • Page 1 of 1

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

Res Sq 2021 May 19. Epub 2021 May 19.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
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http://dx.doi.org/10.21203/rs.3.rs-515215/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142659PMC
May 2021

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

bioRxiv 2021 May 11. Epub 2021 May 11.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
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http://dx.doi.org/10.1101/2021.05.10.443524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132225PMC
May 2021

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

Science 2020 12 5;370(6523):1473-1479. Epub 2020 Nov 5.

Quantitative Biosciences Institute (QBI) Coronavirus Research Group Structural Biology Consortium, University of California, San Francisco, CA, USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.
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http://dx.doi.org/10.1126/science.abe3255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857409PMC
December 2020

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

Science 2020 12 15;370(6521). Epub 2020 Oct 15.

Quantitative Biosciences Institute (QBI) COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
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http://dx.doi.org/10.1126/science.abe9403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808408PMC
December 2020

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation.

bioRxiv 2020 Aug 17. Epub 2020 Aug 17.

Quantitative Biosciences Institute (QBI) Coronavirus Research Group Structural Biology Consortium, University of California, San Francisco, CA, USA.

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.
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http://dx.doi.org/10.1101/2020.08.08.238469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430568PMC
August 2020

Structural basis for distinct operational modes and protease activation in AAA+ protease Lon.

Sci Adv 2020 May 20;6(21):eaba8404. Epub 2020 May 20.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo-electron microscopy to determine structures of the Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, "open" spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed "closed" conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates.
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http://dx.doi.org/10.1126/sciadv.aba8404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239648PMC
May 2020

The molecular principles governing the activity and functional diversity of AAA+ proteins.

Nat Rev Mol Cell Biol 2020 01 21;21(1):43-58. Epub 2019 Nov 21.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.

ATPases associated with diverse cellular activities (AAA+ proteins) are macromolecular machines that convert the chemical energy contained in ATP molecules into powerful mechanical forces to remodel a vast array of cellular substrates, including protein aggregates, macromolecular complexes and polymers. AAA+ proteins have key functionalities encompassing unfolding and disassembly of such substrates in different subcellular localizations and, hence, power a plethora of fundamental cellular processes, including protein quality control, cytoskeleton remodelling and membrane dynamics. Over the past 35 years, many of the key elements required for AAA+ activity have been identified through genetic, biochemical and structural analyses. However, how ATP powers substrate remodelling and whether a shared mechanism underlies the functional diversity of the AAA+ superfamily were uncertain. Advances in cryo-electron microscopy have enabled high-resolution structure determination of AAA+ proteins trapped in the act of processing substrates, revealing a conserved core mechanism of action. It has also become apparent that this common mechanistic principle is structurally adjusted to carry out a diverse array of biological functions. Here, we review how substrate-bound structures of AAA+ proteins have expanded our understanding of ATP-driven protein remodelling.
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http://dx.doi.org/10.1038/s41580-019-0183-6DOI Listing
January 2020

Unique Structural Features of the Mitochondrial AAA+ Protease AFG3L2 Reveal the Molecular Basis for Activity in Health and Disease.

Mol Cell 2019 09 18;75(5):1073-1085.e6. Epub 2019 Jul 18.

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA. Electronic address:

Mitochondrial AAA+ quality-control proteases regulate diverse aspects of mitochondrial biology through specialized protein degradation, but the underlying mechanisms of these enzymes remain poorly defined. The mitochondrial AAA+ protease AFG3L2 is of particular interest, as genetic mutations localized throughout AFG3L2 are linked to diverse neurodegenerative disorders. However, a lack of structural data has limited our understanding of how mutations impact enzymatic function. Here, we used cryoelectron microscopy (cryo-EM) to determine a substrate-bound structure of the catalytic core of human AFG3L2. This structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins. Many disease-relevant mutations localize to these unique structural features of AFG3L2 and distinctly influence its activity and stability. Our results provide a molecular basis for neurological phenotypes associated with different AFG3L2 mutations and establish a structural framework to understand how different members of the AAA+ superfamily achieve specialized biological functions.
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http://dx.doi.org/10.1016/j.molcel.2019.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731152PMC
September 2019

Epitope mapping of diverse influenza Hemagglutinin drug candidates using HDX-MS.

Sci Rep 2019 03 18;9(1):4735. Epub 2019 Mar 18.

Janssen Vaccines and Prevention, Janssen Pharmaceutical Companies of Johnson & Johnson, Archimedesweg 6, 2333 CN, Leiden, The Netherlands.

Epitope characterization is critical for elucidating the mechanism of action of drug candidates. However, traditional high-resolution epitope mapping techniques are not well suited for screening numerous drug candidates recognizing a similar target. Here, we use Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the conformational impact of diverse drug molecules binding on Hemagglutinin (HA), the major surface antigen of influenza viruses. We optimized a semi-automated HDX-MS workflow to systematically probe distantly related HA subtypes in complex with 4 different drug candidates, ranging from a monoclonal antibody to a small synthetic peptide. This fast, cost-effective HDX-MS epitope mapping approach accurately determined the main antigenic site in all cases. Moreover, our studies reveal distinct changes in the local conformational dynamics of HA associated to the molecular mechanism of neutralization, establishing a marker for broad anti-HA activity. Taken together, these findings highlight the potential for HDX-MS epitope mapping-based screening to identify promising candidates against HA at early stages of drug discovery.
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http://dx.doi.org/10.1038/s41598-019-41179-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427009PMC
March 2019

Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing.

Science 2017 11;358(6363)

Department of Integrative Structural and Computational Biology, The Scripps Research Institute HZ 175, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo-electron microscopy structure reveals how the adenosine triphosphatases (ATPases) form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring adenosine triphosphate hydrolysis cycle that results in stepwise substrate translocation. A hingelike linker accommodates the large-scale nucleotide-driven motions of the ATPase spiral relative to the planar proteolytic base. The translocation mechanism is likely conserved for other AAA+ ATPases.
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http://dx.doi.org/10.1126/science.aao0464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829300PMC
November 2017

Reciprocal Degradation of YME1L and OMA1 Adapts Mitochondrial Proteolytic Activity during Stress.

Cell Rep 2016 Mar 25;14(9):2041-2049. Epub 2016 Feb 25.

Department of Molecular and Experimental Medicine, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

The mitochondrial inner membrane proteases YME1L and OMA1 are critical regulators of essential mitochondrial functions, including inner membrane proteostasis maintenance and mitochondrial dynamics. Here, we show that YME1L and OMA1 are reciprocally degraded in response to distinct types of cellular stress. OMA1 is degraded through a YME1L-dependent mechanism in response to toxic insults that depolarize the mitochondrial membrane. Alternatively, insults that depolarize mitochondria and deplete cellular ATP stabilize active OMA1 and promote YME1L degradation. We show that the differential degradation of YME1L and OMA1 alters their proteolytic processing of the dynamin-like GTPase OPA1, a critical regulator of mitochondrial inner membrane morphology, which influences the recovery of tubular mitochondria following membrane-depolarization-induced fragmentation. Our results reveal the differential stress-induced degradation of YME1L and OMA1 as a mechanism for sensitively adapting mitochondrial inner membrane protease activity and function in response to distinct types of cellular insults.
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http://dx.doi.org/10.1016/j.celrep.2016.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785047PMC
March 2016

Affinity Maturation of a Potent Family of HIV Antibodies Is Primarily Focused on Accommodating or Avoiding Glycans.

Immunity 2015 Dec;43(6):1053-63

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Scripps Center for HIV/AIDS Vaccine Immunology & Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

The high-mannose patch on the HIV-1 envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet some variation evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provides insights to advance design of HIV vaccine candidates.
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http://dx.doi.org/10.1016/j.immuni.2015.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692269PMC
December 2015

Contrast sensitivity comparison between AcrySof ReSTOR and Acri.LISA aspheric intraocular lenses.

J Refract Surg 2010 Jul;26(7):471-7

Instituto Oftalmológico Fernández-Vega, Oviedo, Spain.

Purpose: To evaluate distance contrast sensitivity under photopic, mesopic, and mesopic with glare conditions after implantation of the AcrySof ReSTOR (Alcon Laboratories Inc) and Acri.LISA (Carl Zeiss Meditec) intraocular lenses (IOLs).

Methods: Binocular contrast sensitivity function was measured with the Optec 6500 FACT contrast sensitivity chart at distance and at three lighting conditions (85 cd/m2 and 3 cd/m2 with and without glare) in 36 eyes of 18 patients implanted with the AcrySof ReSTOR aspheric SN6AD3 IOL and 40 eyes of 20 patients implanted with the Acri.LISA 366D IOL. Results after implantation were compared between lenses at 1 and 6 months.

Results: Our results revealed that both IOLs provided good best spectacle-corrected visual acuities at distance and near vision (approximately 20/20), and no statistically significant differences were noted between models at different time points after surgery. Both IOLs provided contrast sensitivity within the normal range in photopic conditions. Under low lighting conditions, a reduction in contrast sensitivity for both lenses, particularly at higher spatial frequencies, was noted. No significant differences were observed between both IOLs at any lighting condition.

Conclusions: The AcrySof ReSTOR SN6AD3 and Acri.LISA aspheric IOLs provided contrast sensitivity within normal range under photopic conditions and a reduction in contrast sensitivity under mesopic conditions, with no significant differences between the two brands.
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http://dx.doi.org/10.3928/1081597X-20090728-04DOI Listing
July 2010

Intermediate visual function with different multifocal intraocular lens models.

J Cataract Refract Surg 2010 May;36(5):733-9

Fernández-Vega Ophthalmological Institute, Surgery Department, School of Medicine, University of Oviedo, Oviedo, Spain.

Purpose: To compare visual acuity at different distances after bilateral implantation of 1 of 4 multifocal intraocular lenses (IOLs).

Setting: Fernández-Vega Ophthalmological Institute, Oviedo, Spain.

Methods: This study evaluated consecutive patients who had bilateral implantation of a spherical multifocal IOL with a +4.00 diopter (D) addition (add) (AcrySof ReSTOR SN60D3) or an aspheric multifocal IOL with a +4.00 D add (AcrySof ReSTOR SN6AD3), +3.75 D add (Acri.LISA 366D), or +3.00 D add (AcrySof ReSTOR SN6AD1). Six months postoperatively, binocular measurement of corrected distance visual acuity (CDVA) at 4 m, corrected near visual acuity (CNVA) at 40 cm, and corrected intermediate visual acuity (CIVA) at 50, 60, 70, and 80 cm were performed; the defocus curve was also measured.

Results: Each IOL model was implanted in 20 eyes (10 patients). All IOL models resulted in good distance vision, with no statistically significant differences between models. Patients with the +3.00 aspheric IOL had statistically better binocular CIVA at 50, 60, and 70 cm (P<.0001) and binocular CNVA at 40 cm (P = 3 x 10(-3)) than patients with the other IOL models.

Conclusion: Patients with bilateral multifocal aspheric IOLs with a lower add had better intermediate and distance near visual acuity than patients with bilateral multifocal spherical IOLs or bilateral aspheric IOLs with a higher add.
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http://dx.doi.org/10.1016/j.jcrs.2009.11.018DOI Listing
May 2010

Visual acuity comparison of 2 models of bifocal aspheric intraocular lenses.

J Cataract Refract Surg 2009 Apr;35(4):672-6

Fernández-Vega Ophthalmological Institute, Surgery Department, School of Medicine, University of Oviedo, Oviedo, Spain.

Purpose: To compare visual acuity after bilateral implantation of 2 models of multifocal aspheric intraocular lenses (IOLs).

Setting: Fernández-Vega Ophthalmological Institute, Oviedo, and University of Valencia, Valencia, Spain.

Methods: Prospective study of patients who had bilateral implantation of an AcrySof ReSTOR SN6AD3 IOL (Group 1) or an Acri.LISA 366D IOL (Group 2). Six months postoperatively, binocular uncorrected and best corrected distance visual acuity, uncorrected-distance and best distance-corrected near visual acuity, best corrected intermediate visual acuity, and the defocus curve were measured in both IOL groups.

Results: Group 1 comprised 36 eyes (18 patients) and Group 2, 40 eyes (20 patients). The mean binocular values in Group 1 and Group 2, respectively, were as follows: best corrected distance acuity, -0.05 +/- 0.09 logMAR and -0.08 +/- 0.08 logMAR (both approximately 20/20); best distance-corrected near acuity, -0.01 +/- 0.16 logMAR and -0.05 +/- 0.07 logMAR; best corrected intermediate acuity at 80 cm, 0.20 +/- 0.18 logMAR (approximately 20/32) and 0.16 +/- 0.13 logMAR (approximately 20/25) and at 60 cm, 0.16 +/- 0.16 logMAR (approximately 20/25) and 0.18 +/- 0.13 logMAR (approximately 20/25). There were no statistically significant between-group differences in visual acuity at any distance (P>.3). Defocus curves were similar between groups (2.00 to -5.00 diopters) (P>.26).

Conclusions: The 2 multifocal aspheric IOL models gave similar and good high-contrast visual acuity at distance and near. Intermediate visual acuity, also comparable between IOL models, was better than published results of a spherical IOL model.
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http://dx.doi.org/10.1016/j.jcrs.2008.11.061DOI Listing
April 2009
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