1H, 13C, and 15N resonance assignments of a the amyloidogenic peptide SEM2(49-107) by NMR spectroscopy

doi:10.21203/rs.3.rs-4477618/v1

It has been shown that human seminal fluid is a major factor in enhancing HIV activity. The SEM2(49–107) peptide is a product of cleavage after ejaculation by internal prostheses of the semenogelin 2 protein, expressed in seminal vesicles. It is established that the peptide SEM2(49–107) forms amyloid fibrils, which increase probability of contracting HIV infection. In this nuclear magnetic resonance (NMR) study, we present almost complete (86%) resonance distributions for the ¹H, ¹⁵N and ¹³C atoms of the backbone and side-chain of the SEM2peptide (49–107) (BioMagResBank accession number 52356). The secondary structure of SEM2(49–107) peptide was estimated by using two approaches, secondary chemical shifts analysis (CSI) and TALOS-N prediction. Analysis of the secondary structure of the SEM2(49–107) peptide using both methods revealed that the peptide contains helical segments at the C-terminal. Also in this work, we used phase-sensitive 2D HSQC ¹H-¹⁵N experiments measuring longitudinal T₁ and transverse T₂ NMR relaxation times to report predicted secondary structure and backbone dynamics of the SEM2(49–107) peptide. This resonance assignment will form the basis of future NMR research, contributing to a better understanding of the peptide structure and internal dynamics of molecule.

HIV

Semenogelin-2

SEM2(49-107)

Protein NMR

Resonance assignment

The Human Immunodeficiency Virus (HIV) remains a global public health problem (Deeks et al 2015; Simon 2006). HIV is an infection that affects the body's immune system. The last stage of HIV infection is known as acquired immunodeficiency syndrome (AIDS) (Piot et al 2001). HIV damages white blood cells, that leads to a weakened immune system. This fact increases the probability of developing diseases such as tuberculosis, infectious diseases, and some types of oncology (Dimitrov 1993). The infection can be transmitted through different body fluids of HIV infected people, such as blood, breast milk, and seminal fluid. HIV can also be passed on to a baby during pregnancy. Human semen is the main factor of increasing HIV’s activity. Seminal fluid is considered as the main sexually transmitted vector of the HIV virus. The main component of seminal fluid is a viscous clot composed mainly of sperm cells and the proteins semenogelin 1 and semenogelin 2 (Röcker et al 2005). Activity of the PSA (Prostate-Specific Antigen) seminal protease causes rapid liquefaction of the seminal fluid and the cleavage of the semenogelins into short peptide fragments and then released.

It has been established that the probability of HIV infection increases by 3-5 times with the presence of amyloid fibrils in seminal fluid (Westermark 2005). So-called SEVI (semen-derived enhancers of viral infection) fibrils, which are formed from peptide fragments of prostatic acid phosphatase (PAP): PAP (248-286) and PAP (85-120) (Brender et al 2009), (Blokhin, D. S. 2022). However, additional studies have shown that SEVI are not the only type of amyloid fibrils included in human seminal fluid (Roan 2011). Roan et al discovered new type of semen amyloids, called as SEM amyloid (Roan et al 2014). SEM amyloid precursor proteins are two homologous proteins, SEM1 and SEM2, which are mainly expressed in seminal vesicles and form a major component of the sperm clot (Lilja et al 1989). It was also revealed that semenogelin proteins are rapidly cleaved after ejaculation by internal proteases with subsequent release of 7 short amyloidogenic peptide fragments such as SEM1(45-107), SEM2(45-107), SEM1(49-107), SEM2(49-107), SEM1(68-107), SEM2(68-107) and SEM1(86-107) (Blokhin, D.S. 2021).

The amyloid fibrils of sperm involved in increasing the viral activity of HIV, and currently they actively are studied (Roan et al 2011), (Blokhin, D. S. 2023). However, some questions regarding the structure of fibrils, their functions, and their mechanism of interaction with virions and cells in semen remain unanswered.

In this work, we analyzed one of the peptide fragments semenogelin 2 SEM2(49-107) peptide. Here, we report backbone and side-chain ¹H, ¹⁵N, and ¹³C (NMR) resonance assignment for SEM2(49-107) peptide.

Protein expression and purification

The SEM2(49-107) of Homo sapiens semenogelin 2 gene was amplified from human genomic DNA. Sites of Ndel and Xhol restriction endonucleases as well as stop codon TGA were introduced by constructing following forward and reverse primers SEMG2_59_fwd (ttttttttcatatgTTTGGACAAAAAGACCAACAACATACTAAATCCAAA) and SEMG2_rev (ttttttttctcGAGCAGTTGTTGACTTCCACCTAG) which were purchased by Evrogen (Moscow, Russia) To construct SEM2(49-107) peptide with additional 6×his-tag at the С-terminus, the structural gene sem2.59 was cloned into pGS21a vector. The expression vector was multiplied by transformation into E. coli DH5α strain. Then E. coli strain BL21 star (DE3) pLysS was transformed with SEMG2(59)::pGS21a vector for heterologous expression. Protein expression was performed on selective synthetic minimal medium (M9) containing antibiotics (ampicillin - 100μg/mL; chloramphenicol, 25 μg/mL) with isotope labeled glucose (¹³С) and ammonium chloride (¹⁵N) (Cambridge Isotope Laboratories, UK) as a source of carbon and nitrogen (Usachev 2018). Primarily the cells were grown in LB rich nutrient medium at 37 °C with shaking at the rate of 180 rpm. At optical density OD₆₀₀ equal to 0.9 cells were harvested in sterile environment, washed and transferred into M9 medium. Then we grew the cells with same conditions for 40 min.

The expression of the sem2.59 was induced by adding isopropy-β-D-1-thiogalactopyranoside (IPTG OD₆₀₀=0.6–0.8) to a final concentration of 1 mM. Expression was carried out for 16h at the temperature of 37 °C and shaking rate of 180 rpm. Then cells were collected by centrifugation (5000 rpm, 10 min, 4 °C) using Avanti JXN-26, rotor JLA-9.1000 (Beckman, USA), the pellet was frozen in a liquid nitrogen and stored at –24 °C.

The cells were disrupted at 4 °C in buffer 1 (50 mM Tris-HCl, pH 7.6, 0.3 M NaCl) with a HD2070 ultrasonic homogenizer (Bandelin, Germany) in the presence of protease inhibitor cocktail (Mini Protease Inhibitor Cocktail (Roche, Switzerland)). Cell debris was pelleted by centrifugation at 100,000g for 1 h at 4°C using Optima XPN (45Ti rotor) centrifuge (Beckman Coulter, USA). The supernatant was discarded, and the precipitate with the target protein in the inclusion bodies was used for refolding. For further extraction of SEM2.59 protein, the precipitate was resuspended in buffer 2 (50 mM Tris-HCl, pH 7.6, 0.3 M NaCl, 1M Urea) using an ultrasonic homogenizer, centrifuged at 27000g for 40 min at 4°C, the supernatant was drained, and the precipitate was repeated twice more. After the third centrifugation, the precipitate was left to dissolve overnight on a magnetic stirrer at 4°C in a solution of 8M Urea. The resulting mixture was applied to a NiNTA resin column equilibrated with 8M Urea solution. Chelate affinity chromatography (IMAC) was performed on NiNTA resin followed by elution of SEM2.59 protein with buffer 3 (8M Urea; 300 mM imidazole), fractions of 0.5 mL each.Gel filtration was performed using an NGS Discover chromatographic system and Enrich SEC70 column (BioRad, USA) in buffer 4 (25 mM Bis-Tris pH 6.0, 0.2 % SDS).

NMR spectroscopy

SEM2(49-107) is a peptide of 59 amino acid residues corresponding to 49-107 amino acid residues of the human semenogelin-2 protein. Amino acid sequence of SEM2(49-107) peptide: MFGQKDQQHTKSKGSFSIQHTYHVDINDHDWTRKSQQYDLNALHKATKSKQHLGGSQQLLEHHHHH. The SEM2(49-107) construct used in this study comprises 59 amino acids including 6 histidine residues (6 × his tag) at the C-terminus to aid in protein purification using affinity chromatography. The 1D (1H), 2D (1H-15N) and 3D (¹H-¹³C-¹⁵N) NMR spectra of the SEM2 (49-107) peptide (C=0.46mM, H₂O+D₂O/ 90%+10%) were carried out on NMR spectrometer with a 700 MHz frequency (Bruker, AVANCE III 700) equipped with a quadruple resonance (1H, 13C, 15N, 31P) CryoProbe at temperature of 293K. Data processing was performed using the Bruker Topspin 3.6 software. All spectra were analyzed in the CCP-NMR program (Vranken, W.F. 2005). During the experiments, no changes were observed in the 1H NMR spectra of the peptide SEM2 (49-107). Based on this, it is assumed that there have been no structural changes in the peptide.

The 2D ¹H-¹⁵N HSQC experiment was used to determine the chemical shifts of the nitrogen ¹⁵N signals and assign their spin systems.

The following 3D (¹H-¹³C-¹⁵N) NMR experiments were performed to reconstruct the amino acid sequences: HNCO, HN(CO)CA, HNCA, HN(CA)CO, HN(CO)CACB, HNCACB, HN(CO)CC, ¹⁵N-¹H HSQC-TOCSY (0.12s mixing time) and ¹⁵N-¹H HSQC-NOESY (0.1 s mixing time), to find the HN, N, CO signals of the main chain, and the Ca, Cb, Ha, Hb and others signals of the side chains. The list of recorded NMR spectra is provided in Table 1. The obtained values of chemical shifts for various amino acids were compared to the literature data (https://bmrb.io/ref_info/csstats.php). Spectra for the measurement of longitudinal (R₁) and transverse (R₂) relaxation rates of the ¹⁵N amide nuclei and ¹⁵N-¹H heteronuclear NOE values were collected on NMR spectrometer with a 700 MHz frequency at 293K.

Assignments and data deposition

The 2D ¹H–¹⁵N HSQC NMR spectrum of SEM2(49-107) peptide with resonance assignments are presented in Fig. 1. This experiment was used to determine the chemical shifts of the nitrogen ¹⁵N signals and assign their spin systems. We showed the labeled ¹H-¹⁵N HSQC spectrum for SEM2(49-107) peptide in Fig. 1, while the residues in His-tag were not assigned in this figure. The unassigned/unpicked resonances in Fig. 1 were from NH₂ region and 6 histidine residues (6 × his tag) at the C-terminus of amino acids.

Backbone and side-chain resonance assignments of SEM2(49-107) peptide were obtained based on the conventional multi-dimensional experiments. The backbone data was obtained from HNCO, HN(CA)CO, HN(CO)CA, HNCA, HN(CO)CACB, HNCACB spectra. Side-chain resonance assignment of SEM2(49-107) was carried out using HNCACB, HBHACONH, ¹⁵N-¹H HSQC-TOCSY, ¹⁵N-¹H HSQC-NOESY and HC(CO)NH experiments. We assigned most of backbone and side-chain resonances of the SEM2(49-107) peptide, including 82.8% of HN, 82.8% of ¹⁵N, 81.4% of ¹³Cα, 76.3% of ¹³Cβ, 76.3% of ¹Hα, 62.7% of ¹Hβ and 83.1% of ¹³C chemical shifts.

Standard triple resonance assignment of protein backbone is based on HNCACB and HN(CO)CACB spectra (Schmieder 2006). The idea is that the CBCANH correlates each NH group with the Cα and Cβ chemical shifts of its own residue (strongly) and of the previous residue (weakly) (Sanchugova 2021). By superimposing the HNCACB spectrum on HN(CO)CACB, signals belonging to Cαi-1 and Cβi-1 of the previous residue can be identified. The HNCA and HN(CO)CA experiments give the same information as the HNCACB and HN(CO)CACB expect for the absence of Cβ resonance.

The ¹H, ¹⁵N, and ¹³C chemical shift values for SEM2(49-107) have been deposited at the BioMagResBank (http://www.bmrb.wisc.edu/) under BMRB accession number 52356. Most of the unassigned residues were in the central region of SEM2(49-107) and showed severe overlapping, broadening or were otherwise difficult to distinguish from noise signals in the spectra. The spectra did not contain signals of amide proton HN in the center of the chain between 72 (Asp) and 78 (Thr) amino acids, 86 (Asp) and 88 (Asn). This may be due to their high conformational mobility and rapid chemical exchange of amide protons HN and deuterium of the solvent for these residues.

Table 1. Acquisition parameters used in the assignment experiments

№	Experiment	Time domain data size (points)			Spectral width (ppm)			ns	Delay time [s]
№	Experiment	T₁	T₂	T₃	F₁	F₂	F₃
1	¹H-¹⁵N HSQC	512	2048	-	12 (¹H)	40 (¹⁵N)	-	2	1.6
2	HNCO	88	48	2048	22 (¹³С)	36 (¹⁵N)	15 (¹H)	8	1
3	HN(CA)CO	128	48	2048	22 (¹³C)	36 (¹⁵N)	15 (¹H)	16	1
4	HNCA	128	52	2048	50 (¹³C)	35 (¹⁵N)	13 (¹H)	8	1
5	HNCACB	128	48	2048	75 (¹³C)	35 (¹⁵N)	15 (¹H)	16	1
6	HN(CO)CACB	128	64	2048	80 (¹³C)	35 (¹⁵N)	14 (¹H)	8	1
7	HN(CO)HCC	128	56	2048	12 (¹H)	30 (¹⁵N)	12 (¹H)	16	1.2
8	HN(CO)HAHB	128	80	2048	14 (¹H)	31 (¹⁵N)	14 (¹H)	12	1.4
9	HNHAHB	128	64	2048	14 (¹H)	31 (¹⁵N)	14 (¹H)	12	1.6
10	HNHA	64	96	2048	12 (¹H)	12 (¹⁵N)	34 (¹H)	16	1.2
11	¹³C-NOESY-HSQC	256	48	2048	12 (¹H)	70 (¹³C)	12 (¹H)	8	1.2
12	¹⁵N-HSQC-TOCSY	164	48	2048	12 (¹H)	34 (¹⁵N)	12 (¹H)	8	1.2
13	¹H-¹⁵N HSQC for T2	512	2048	-	12 (¹H)	34 (¹⁵N)	-	2	2.0
14	¹H-¹⁵N HSQC for T1	512	2048	-	12 (¹H)	34 (¹⁵N)	-	2	2.0
15	HetNOE (¹H-¹⁵N HSQC-NOE)	512	2048	-	12 (¹H)	34 (¹⁵N)	12	8	3.5

The secondary structure of SEM2(49-107) peptide was valued by using two approaches, secondary chemical shifts analysis (CSI) (Wishart, D. S.1992) and TALOS-N (Shen, Y. 2009) prediction (Fig. 3 and Fig. 4(a) and 4(b). Secondary chemical shifts are obtained by calculating the differences between the assigned chemical shifts and the theoretical random coil chemical shift (Fig 2). According to the CSI (chemical shift index) values, a sequence of three or more consecutive "-1" is referred to as an α-helical element. A sequence consisting of three or more consecutive "1", on the other hand, is termed a β-sheet.

To calculate secondary chemical shifts, one typically compares the assigned chemical shifts of a protein to the corresponding theoretical chemical shifts expected for a random coil conformation. The value of these secondary chemical shifts for H_α, C_α, C_β and C’ (Δδ¹³C_α, Δδ¹³C_β, Δδ¹³C’, Δδ¹³H_α is related to the secondary structure of the individual residues. Positive values of Δδ¹³ Cα and Δδ¹³ correspond to a a-helical conformation whereas negative values correspond to β - sheet structure. For Δδ¹³C_βpositive values reflect β-sheet structure and negative values α-helical conformation (Jacobsen 2022) (Fig.3).

By using the consensus chemical shift index from all spectra mentioned above we found that SEM2 (49-107) peptide contains alpha-helixes fragments at the C-terminus.

The secondary structure elements were predicted by the TALOS-N webserver (Shen 2013) using Cα, Cβ, CO, N and HN chemical shift values of SEM2(49-107) peptide. Figure 4a represents dihedral angles of SEM2(49-107) peptide and Fig. 4b depicts the α-helices and random coil probability predicted by TALOS-N (Maksimov 2021).

Relaxation measurements and analysis

In NMR experiments we determined the relaxation parameters of 15 N nuclei at a temperature of 293 K. We determined the rates of longitudinal relaxation (R₁) and transverse relaxation (R₂) and the magnitude of heteronuclear Overhauser effects ¹⁵N{¹H}.

To assess the mobility of the SEM2 (49-107) peptide, a series of NMR relaxation experiments were performed. Specifically, a phase-sensitive 2D ¹H-¹⁵N HSQC experiment was used to measure relaxation times T₁ and T₂ at various delay times. The relaxation delays to measure R₁ were 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.2, 1.6, and 2.0 s; and to measure R₂ the values were 0, 16, 32, 48, 64, 96, 128, 160, 192 and 256 ms. Based on these experiments, values for the longitudinal and transverse relaxation rates R₁ and R₂ were determined (Figure 5).

The observed difference in the R₂/R₁ rate values suggests that there is a variation in mobility and conformational dynamics along the peptide sequence. Specifically, it indicates that the C-terminus of the peptide has less mobility compared to the N-terminus. In addition, the R₂/R₁ rate values indicate that there is secondary structure in the form of an alpha helix within the amino acid range of 89 (Ala) to 98 (Gln). This suggests that this region of the peptide adopts a relatively constrained conformation, which is consistent with the results of the secondary structure analysis.

The correlation time (τ_c) is used to estimate the dynamics of the SEM2(49-107) peptide. This parameter of τ_c shows the time it takes for a protein molecule to rotate by one radian in solution (Rossi et al., 2010). For the SEM2 (49-107) peptide, an average correlation time τ_c=9.1 ns was calculated. This value may indicate that the protein exists in a monomeric state. The results of the hetNOE ¹⁵N{¹H} experiment is consistent with the NMR experiments for measuring the rate of R₁ and R₂ relaxation. In the NMR experiments, the average hetNOE value was approximately 0.5-0.6.

In this paper we present information about the chemical shifts of SEM2(49-107) peptide. It was possible to identify 48 of the 59 amino acids resides (86%). Based on the analysis of these chemical shifts and predictions of secondary structure according TALOS-N and NMR relaxation (R₁, R₂, hetNOE) the SEM2 peptide (49-107) peptide contains helical fragments (89 (Ala) to 98 (Gln)). in the form of an alpha helix at the C-terminus. The results of both analysis methods show the location of the secondary structure elements.

• NOE

• Nuclear Overhauser effect

• NMR

• nuclear magnetic resonance

• HIV

• human immunodeficiency virus

• SEVI

Semen-derived Enhancer of Viral Infection

• R1

Longitudinal or spin-lattice relaxation rate

• R2

Transverse or spin-spin relaxation rate

Author Contribution

D.B. initiated the project and designed the experimental protocol. D. B. and A. T. performed NMR experiments and resonance assignments, deposited the data to the BMRB, wrote the majority of the manuscript and made the remaining figures. A.B., E. K. expressed and purified protein and prepared samples. V. K. Formal Analysis. All authors reviewed the manuscript.

Acknowledgement

This work is supported by the Russian Science Foundation (project no. 20-73-10034). The NMR study was financially supported by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (project FZSM-2023-0012).

Data Availability

https://bmrb.io/data_library/summary/index.php?bmrbId=52356

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No competing interests reported.

1H, 13C, and 15N resonance assignments of a the amyloidogenic peptide SEM2(49-107) by NMR spectroscopy

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Abstract

Figures

Biological context

Methods and experiments

Conclusions

Abbreviations

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

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