Biology subjects

Almeida, F. C. L.

Publications and source records attributed to Almeida, F. C. L..

6 recordsLinked to original sources

bioRxiv2022

Structural and dynamical basis for the interaction of HSP70-EEVD with JDP Sis1

We employed NMR spectroscopy to investigate the structure and dynamics of the class B J domain protein (JDP) of S. cerevisiae (Sis1) complexed with an EEVD peptide of HSP70. It is widely recognized that the interactions between the EEVD motif and Sis1 play a crucial role in the chaperone activity. Notably, the deletion of the EEVD impairs the ability of Sis1 to bind with HSP70, while leaving the interaction between the class A JDP Ydj1 and HSP70 unaffected. Leveraging the advantages of NMR, which is particularly suitable for studying transient interactions, we provide compelling evidence that the EEVD motif transiently engages multiple sites on Sis1. Our findings revealed that EEVD binds to two distinct sites within the C-terminal domain I (CTDI) of Sis1. The interaction at these sites plays a crucial role in anchoring HSP70 to Sis1 at site I, as well as displacing the client protein at site II. Notably, site II is also the binding site for the client protein, and its displacement occurs through competition with the binding to site II. In addition to these interactions, we observed that EEVD, as a transient electrostatic binder, also interacts with the J domain and the GF-rich loop located between the J domain and -helix 6. We propose that the interaction between EEVD and Sis1 facilitates the dissociation of -helix 6, promoting a conformational state that is more favorable for interaction with HSP70 at the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) interface. We also employed -synuclein as a substrate to investigate the competitive nature between EEVD and the client protein. Our experimental findings provided evidence supporting the interaction of EEVD with the client protein at multiple sites. Our findings contribute essential insights into the mechanistic cycle of class B JDPs, paving the way toward a more complete understanding of the primary function of Sis1, which is the transfer of the client protein to HSP70, where multiple site transient interactions play a collective role.

molecular biology↗

bioRxiv2021

Detection of Selenium/Sulphur substitution in the heterologous expression of Thioredoxin isoform 1 (Trx1) from Saccharomyces cerevisiae.

Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous translation. Furthermore, the Maldi-TOF spectra are showing the presence of the most abundant isotopes of selenium inserted in the peptides containing cysteine and methionine, derived from the Trx1 digestion. The one dimensional 77Se-1H heteronuclear multiple quantum coherence NMR spectroscopy (1D-HMQC) for reduced Seleno substituted Trx1 (Se-Trx1), revealed three ressonance lines for 1H{beta}1 from Selenocysteines 30 and 33, between 1.6 and 2,0 ppm. The bidimensional HMQC spectra (2D-HMQC) of the reduced Se-Trx1 show the 77 Se ressonance signal in 178 ppm, coupled with 1H{beta}1 and 1H{beta}2 lines between 2.1 and 1.8 ppm. The 1D-HMQC for oxidized Trx1 revealed the only one broad resonance in 2.6 ppm probably relative to the 1H{beta}1 protons. The 2D-HMQC spectrum of oxidized protein shows a higher chemical shift of selenocysteine 77Se (832 ppm) if compared to reduced state (178 ppm). Together these data are showing that the protocol of Se - S substitution developed here is a efficient method to label the active site of Thioredoxin 1 with a broad band chemical shift atom 77Se. Furthermore the large spectral window of the 77Se NMR detected between reduced and oxidized states of the Thioredoxin 1 shows that this atom is an excellent probe for accessing oxidative states and probably the conformational dynamics of the active site of the Se-Trx1.

biochemistry↗

bioRxiv2021

Structure insights, thermodynamic profiles, dsDNA melting activity, and liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid N-terminal domain binding to DNA

The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD), either with or without the SR-rich motif (SR), upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on specificity for N-NTD/N-NTD-SR interaction with TRS, including an unfavorable energetic contribution to binding along with hydrogen bonds between the triple-thymidine (TTT) motif in the dsTRS and {beta}-sheet II due to the defined position and orientation of the DNA duplex, a well-defined pattern ({Delta}H > 0 and {Delta}S > 0 for ssTRS, and {Delta}H < 0 and {Delta}S < 0 for dsTRS) for the thermodynamic profile of binding, and a preference for TRS in the formation of liquid condensates when compared to a non-specific sequence. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.

biophysics↗

bioRxiv2021

A systematic structural comparison of all solved small proteins (4-6 kDa) reveals the weight of disulfide bonds in proteins' foldability

Defensins are small proteins, usually ranging from 4 to 6 kDa, amphipathic, disulfide-rich, and with a small or even absent hydrophobic core. Since a hydrophobic core is generally found in globular proteins that fold in an aqueous solvent, the peculiar fold of defensins can challenge tertiary protein structure predictors. We performed a PDB-wide survey of small proteins (4-6 kDa) to understand the similarities of defensins with other small disulfide-rich proteins. We found no differences when we compared defensins with non-defensins regarding the proportion and exposition to the solvent of apolar, polar, and charged residues. Then we divided all small proteins (4-6 kDa) deposited in PDB into two groups, one group with at least one disulfide bond (bonded, defensins included) and another group without any disulfide bond (unbonded). The group of bonded proteins presented apolar residues more exposed to the solvent than the unbonded group. The ab initio algorithm for tertiary protein structure prediction Robetta was more accurate to predict unbonded than bonded proteins. Our work highlights one more layer of complexity for the tertiary protein prediction structure: small disulfide-rich proteins ability to fold even with a poor hydrophobic core.

biochemistry↗

bioRxiv2020

Dynamics of the N-terminal domain of SARS-CoV-2 nucleocapsid protein drives dsRNA melting in a counterintuitive tweezer-like mechanism

The N protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. Its N-terminal domain (NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, initiating melting. N-NTD dsRNA destabilizing activity was more efficient for dsTRS than dsNS. N-NTD dynamics, especially a tweezer-like motion of {beta}2-{beta}3 and 2-{beta}5 loops, played a key role in WC base-pairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD:dsRNA), matching MD simulations and raising different possibilities for N-NTD action: (i) two N-NTDs of dimeric N would act independently, increasing efficiency; (ii) two N-NTDs of dimeric N would bind to two different RNA sites, bridging distant regions of the genome; and (iii) monomeric N would be active, opening up the possibility of a regulatory dissociation event. IMPORTANCECoronaviruses are among the largest positive-sense RNA viruses. They display a unique discontinous transcription mechanism, involving N protein as a major player. The N-NTD promote the dsRNA melting releasing the nascent sense negative strand via a poorly known mechanism of action. It specifically recognizes the body TRS conserved RNA motif located at the 5 end of each ORF. N protein has the ability to transfer the nascent RNA strand to the leader TRS. The mechanism is essential and one single mutation at the RNA binding site of the N-NTD impairs the viral replication. Here, we describe a counterintuitive mechanism of action of N-NTD based on molecular dynamics simulation and kinetic modelling of the experimental melting activity of N-NTD. This data impacts directly in the understanding of the way N protein acts in the cell and will guide future experiments.

biophysics↗

bioRxiv2020

Biophysical and dynamic characterization of a fine-tuned binding of the human Respiratory Syncytial Virus M2-1 core domain to long RNAs

The human Respiratory Syncytial Virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here it is presented the first evidence that hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs, as well as a biophysical and dynamic characterization of the cdM2-1/RNA complex. The main contact region of cdM2-1 with RNA was the 1-2-5-6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their terminals over the domain. The 2-3 and 3-4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to unraveling interaction aspects necessary to M2-1 activity. IMPORTANCEThe main outcome is the molecular description of a fine-tuned binding of the cdM2-1/RNA complex and the evidence that the domain alone has an unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Orthopneumovirus, as the human Respiratory Syncytial Virus (hRSV), stands out for the unique role of M2-1 as a transcriptional antitermination factor able to increase the RNA polymerase processivity.

biophysics↗