The response of T4 lysozyme to large-to-small substitutions within the core and its relation to the hydrophobic effect.

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Waal''s interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu --> Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).     

Biallelic SUN5 Mutations Cause Autosomal-Recessive Acephalic Spermatozoa Syndrome

Acephalic spermatozoa syndrome is a rare and severe form of teratozoospermia characterized by a predominance of headless spermatozoa in the ejaculate. Family clustering and consanguinity suggest a genetic origin; however, causative mutations have yet to be identified. We performed whole-exome sequencing in two unrelated infertile men and subsequent variant filtering identified one homozygous (c.824C>T [p.Thr275Met]) and one compound heterozygous (c.1006C>T [p.Arg356Cys] and c.485T>A [p.Met162Lys]) SUN5 (also named TSARG4) variants. Sanger sequencing of SUN5 in 15 additional unrelated infertile men revealed four compound heterozygous (c.381delA [p.Val128Serfs^∗7] and c.824C>T [p.Thr275Met]; c.381delA [p.Val128Serfs^∗7] and c.781G>A [p.Val261Met]; c.216G>A [p.Trp72^∗] and c.1043A>T [p.Asn348Ile]; c.425+1G>A/c.1043A>T [p.Asn348Ile]) and two homozygous (c.851C>G [p.Ser284^∗]; c.350G>A [p.Gly114Arg]) variants in six individuals. These 10 SUN5 variants were found in 8 of 17 unrelated men, explaining the genetic defect in 47.06% of the affected individuals in our cohort. These variants were absent in 100 fertile population-matched control individuals. SUN5 variants lead to absent, significantly reduced, or truncated SUN5, and certain variants altered SUN5 distribution in the head-tail junction of the sperm. In summary, these results demonstrate that biallelic SUN5 mutations cause male infertility due to autosomal-recessive acephalic spermatozoa syndrome.