key: cord-0742158-yrj6fswd
authors: Das, Anirban; Vishvakarma, Vicky; Dey, Arpan; Dey, Simli; Gupta, Ankur; Das, Mitradip; Vishwakarma, Krishna Kant; Roy, Debsankar Saha; Yadav, Swati; Kesarwani, Shubham; Venkatramani, Ravindra; Maiti, Sudipta
title: Biophysical Properties of the Isolated Spike Protein Binding Helix of Human ACE2
date: 2021-06-30
journal: Biophys J
DOI: 10.1016/j.bpj.2021.06.017
sha: 14dc78655e29dd254e0243bddda411ddab7412f3
doc_id: 742158
cord_uid: yrj6fswd

The entry of the SARS-CoV2 virus in human cells is mediated by the binding of its surface spike protein to the human Angiotensin-Converting Enzyme 2 (ACE2) receptor. A 23 residues long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore, has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain (RBD) of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. While SBP1 shows good solubility (solubility > 0.8 mM), CD spectroscopy shows that it is mostly disordered with some anti-parallel beta-sheet content, and no helicity. The helicity is substantial (> 20%) only upon adding high concentrations (≥ 20% v/v) of 2,2,2-trifluoroethanol, a helix-promoter. Fluorescence correlation spectroscopy and single molecule photobleaching studies show that the peptide oligomerizes at concentrations > 50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1 which do not directly interact with the spike protein to alanine would reduce peptide oligomerization without affecting its spike binding affinity. While the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts so far to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity, but also due to the heretofore unrecognized problem of oligomerization.

them to oligomerize. Efforts to replicate the natural spike protein:ACE2 interface need to 48 focus on both these properties in order to succeed. The primary entry pathway of the SARS-CoV2 virus into the human cell is through the 53 interactions of one of its envelope proteins (the 'Spike' protein), and the human ACE2 54 membrane receptor 1-3 . In high-resolution structures obtained using cryo-EM 4-6 and 55 crystallography 7,8 , it is apparent (Fig. 1A ) that these two proteins predominantly interact via 56 the receptor-binding domain (RBD) of the spike protein and the residues 21-43 from the N-57 terminal -helix of ACE2. This helical segment (called the spike binding peptide or SBP1 58 henceforth) has been of considerable interest to the scientific community, as hypothetically, 59 the peptide segment in its isolated form may competitively bind to the spike RBD and 60 neutralise the virus to protect against COVID infection 9,10 . Additionally, it may be possible to 61 use SBP1 as a recognition element for detection purposes. Indeed, artificial proteins which 62 have been designed based on this helix have displayed affinities (100 pM to 10 nM) 11 which 63 are much greater than that of the original ACE2 protein (15 nM) 6 . Similarly, large 64 immunoadhesins composed of the entire ACE2 12 have been successful in binding to RBD and covalently attaching the 5-(and-6)-carboxy-rhodamine 110, succinimidyl ester (mixed 118 isomers, 5(6)-CR110, SE) dye to the N-terminal free amine of SBP1 and SBP1 mod peptides. 119 The labelling was done on the peptides attached to the resins and the excess dye was 120 thoroughly washed with DMF afterwards. The 5(6)-CR110, SE dye was purchased from 121 Thermo Fisher Scientific (Waltham, Massachusetts, United States). The Cyanine 3 labelled 122 SBP1 (Cy3-SBP1) peptide was similarly synthesized by attaching Cyanine 3 NHS ester dye 123 (Lumiprobe, Maryland, USA) to the N-terminal of the peptide. MALDI-TOF and ESIMS mass 124 spectra of the unlabelled and N-terminal Cy3-labelled peptides are shown in the SI, Figure   125 S1-S4. It is clear from the mass spectra that the dye labelling was nearly complete (∼100%). The supernatant containing the secreted RBD protein was concentrated to 30 ml 141 using a 10 kDa Vivaflow concentrator (Sartorius). The concentrate was diluted with 200 ml 142 of wash buffer containing 35 mM Tris pH 8, 100 mM NaCl, 5% Glycerol, 1 mM PMSF, 1 mM 143 DTT, and concentrated again to 50 ml. The buffer exchanged supernatant was then added to Briefly, a 488 nm laser beam was expanded and collimated using a 1:4 telescope set up 157 before focusing into the sample using an apochromatic 60 water immersion objective with 158 the numerical aperture of 1.2 (Olympus, PA, USA). The fluorescence was collected using the 159 same objective, and focused onto a 15 µm core-diameter optical fiber after filtering through 160 a suitable emission filter (Chroma, VT, USA). The fiber was used as a confocal pinhole to 161 reject the out of focus fluorescence. The fluorescence was detected by a single-photon 162 avalanche photodiode (APD, PerkinElmer Inc., Waltham, MA, USA) and the data were 163 collected and processed using a hardware correlator (PicoHarp 300, PicoQuant, Germany). 164 FCS data were fitted with Equation-1 in Origin 6 software (OriginLab, Northampton, MA, 165 USA). 166 To measure RBD binding, the stock concentration of both the peptides, Rhodamine The average hydrodynamic radii of the peptides were also measured using FCS. considerably helical only when the TFE concentration is 20% (v/v) or higher ( Fig. 2B and 2D ). 298 It is interesting to note that despite the greater helix forming propensities expected for 299 alanine relative to phenylalanine 14 , SBP1 has higher helical content than SBP1 mod in the 300 presence of TFE. hydrodynamic radii (R h ) > 2.5 nm (Fig. 3A, S5 and 3C ). Interestingly, SBP1 had an R h of 3.1 ± 309 0.2 nm, which is larger than that of the SBP1 mod peptide (2.6 ± 0.1 nm). After 24 hours of 310 incubation, both the peptides had a very similar R h of ∼2.5 nm (Fig. 3C) Fig. S6 ). 328 We also investigated whether the fluorescence label has a substantial role in the 329 oligomerization process. We carried out dynamic light scattering (DLS) measurements (using The stoichiometry of the initial oligomers, and the presumed monomers after 341 dissociation, can be directly observed using single molecule photobleaching measurement 342 (smPB) 28, 30, 38 . We performed smPB on two different samples: (1) a freshly diluted one and 343 (2) a sample which is incubated for 24 hours at a concentration of 1 nM. Samples of Cy3-344 labelled SBP1 are subjected to smPB measurements using a total internal reflection 345 fluorescence microscope ( Fig. S8 and S9 , in the SI), as described in the methods section. We 346 observe a distribution of sizes in both the cases (Fig. 4A and 4B) . However, the population at as shown in Fig. 4C and 4D . After the pre-bleaching correction, the stoichiometry at the 355 initial time has a mean of 1.9 ± 0.3, while that after the 24 hours of incubation has a mean 356 of 1.4 ± 0.1. We note that we have not corrected for dissociation that may occur within a 357 short time (~5 minutes) of sample preparation. We also note that the R h observed in FCS for 358 the SBP1 mod is slightly higher than that of SBP1, while the single molecule data shows a 359 similar or lower oligomer stoichiometry [ Fig. S10 ]. This is possibly due to a difference in 360 shape between the two. We also cannot rule out small differences caused by the difference 361 in the fluorescence labels used in the two experiments. little helicity is observed for this isolated peptide. Analyses of our CD data ( Fig. 2A to 2D ) 382 show that the helicity is less than 1%. There is considerable turn and some beta-sheet 383 structure, which suggests that this peptide takes an alternative form when isolated. 384 However, such a secondary structure is rather unlikely to form a binding interface with the What is also notable is that the helix promoter TFE is only moderately effective in promoting 390 helicity within SBP1, as the peptide becomes strongly helical only at TFE concentrations 391 exceeding 20%. 392 A peptide that does not fold into a well-defined secondary structure can be 393 aggregation-prone. However, the solubility of SBP1 is reasonably high, which suggests that 394 the peptide has the means to stabilize its structure, though a non-native one. Probing the 395 size (hydrodynamic radius) of the peptide using FCS yields a hydrodynamic radius of 2.5 nm 396 ( Fig. 3A and 3C) . We note that if the helix retained its structure, the expected hydrodynamic concentrations, is a mixture of monomer, dimer, trimer and even higher oligomers (Fig. 4C) . 411 The solution after a 24 hr incubation period is still not monomeric, but it is considerably 412 enriched with the monomeric species (Fig. 4D) . We note that oligomerization is a 413 concentration-driven process. The relative fraction of monomers is substantial only at low 414 nM concentrations, and would be progressively smaller at higher concentrations. So at µM 415 levels, which will be appropriate for a diagnostic sample, there would be a negligible 416 fraction of monomers available for binding to the RBD. This is what is shown by our RBD 417 binding experiments ( Fig. 1D and 1E ). 418 We reasoned that oligomerization can be a factor in stabilizing a random 419 conformation. An inspection of the peptide bound to the RBD (Fig. 1A and 1B) shows that 420 the surface of the helix that is on the opposite side from the binding interface has three 421 phenylalanine residues, which can be expected to make it prone to form oligomers. We 422 mutated these residues with alanine, which are known to promote helix formation 14 and are 423 not very hydrophobic, to create the variant peptide SBP1 mod (Fig. 1C) . However, the results 424 remain qualitatively similar for the new peptide. The helicity of SBP1 mod is only marginally 425 higher than that of SBP1 (in buffer). Further, SBP1 mod still forms oligomers, though of a 426 smaller size at the higher concentrations and of a somewhat higher size at lower 427 concentrations (Fig. 3C, 3D and S5, in the SI). 428 Overall, our results indicate that designs of high-affinity spike RBD binders based on 429 the SBP1 peptide must overcome at least two major hurdles. These are the lack of helicity of 430 J o u r n a l P r e -p r o o f the SBP1 peptide and its tendency to oligomerize in solution. Also, simple alterations of the 431 sequence are unlikely to make this peptide change these fundamental attributes. Indeed, in 432 recent reports 39 yet to be peer-reviewed, the lack of helicity of SBP1 has been tackled using 433 chemical staples. Nevertheless, the binding affinity of such modified peptides to the spike 434 remains low. Our results suggest that this may be due to the oligomerization of the peptide, 435 which has not been accounted for in all such approaches so far. If the goal is to design a 436 short SBP1-based peptide as a spike protein binder, one must stabilize both the helical 437 secondary structure and the monomeric state of the peptide. 

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A single molecule photobleaching study shows the population distribution of Cy3-666 labelled SBP1 oligomers (A) at time zero and (B) after 24 hours incubation. (C) and (D) are 667 the same as (A) and (B) respectively, but after pre-bleaching correction 38 . The errors are 668 plotted as the standard error of means. For fresh SBP1, n = 323 points (3 sets)